WO2014141386A1 - Two-dimensional cell array device and apparatus for gene quantification and sequence analysis - Google Patents

Two-dimensional cell array device and apparatus for gene quantification and sequence analysis Download PDF

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WO2014141386A1
WO2014141386A1 PCT/JP2013/056818 JP2013056818W WO2014141386A1 WO 2014141386 A1 WO2014141386 A1 WO 2014141386A1 JP 2013056818 W JP2013056818 W JP 2013056818W WO 2014141386 A1 WO2014141386 A1 WO 2014141386A1
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nucleic acid
cell
trapping
extraction device
cells
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PCT/JP2013/056818
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French (fr)
Japanese (ja)
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白井　正敬
神原　秀記
妃代美 谷口
麻衣子 田邉
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株式会社日立製作所
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Priority to CN201380073998.1A priority Critical patent/CN105026562B/en
Priority to JP2015505127A priority patent/JP6093436B2/en
Priority to EP13878277.6A priority patent/EP2975123B1/en
Priority to US14/771,339 priority patent/US10030240B2/en
Priority to PCT/JP2013/056818 priority patent/WO2014141386A1/en
Publication of WO2014141386A1 publication Critical patent/WO2014141386A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow

Definitions

  • the present invention relates to gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, drug discovery, and the like. Specifically, it relates to an mRNA analysis method at the level of one cell.
  • mRNA is extracted from a group of cells, a complementary strand cDNA is prepared, its copy number is amplified by PCR, etc., and the target is placed at the corresponding probe position using a DNA probe array (DNA chip).
  • a method of capturing and detecting fluorescence is used.
  • methods using PCR amplification and DNA chips have low quantitative analysis accuracy, and a highly accurate gene expression profile analysis method has been desired.
  • Quantitative PCR is an analytical method with good quantitativeness.
  • the target is a single cell, the number of mRNA originally present is small and difficult to quantitatively analyze.
  • the sample is divided and quantitative analysis is performed independently, so if there are many genes of interest and genes with low expression levels are included, In some cases, it cannot be measured.
  • Non-patent Document 1 a method for converting all mRNAs into cDNAs and preparing a cDNA library (cDNA aggregate containing all cDNAs) held on beads for use in quantitative analysis. There, it was shown that the expression level of a plurality of genes contained in one cell can be accurately measured by eliminating a measurement error of a small amount of expressed gene by dividing a sample by repeatedly using a cDNA library.
  • FIG. 1 in Non-Patent Document 2 describes a device structure diagram.
  • the chip described here has 300 unit structures arranged so that 300 cells can be processed simultaneously. 3 ⁇ 50 unit structures are arranged on the chip.
  • the unit structure is horizontally long, and the sample solution flows in the longitudinal direction and sequentially reacts while flowing.
  • the reaction volume is 10 nL during the reverse transcription reaction and 50 nL during the PCR reaction, and the volume is reduced.
  • Patent Document 1 discloses a method of constructing a cDNA library using a porous membrane or the like instead of beads. This method uses a device that can obtain a two-dimensional distribution of gene expression and realize gene expression analysis in a large number of cells. When analyzing the expression of a gene in a single cell using this device, it is not necessary to isolate the cell, and mRNA can be directly extracted from a cell in a section of a living tissue and the gene expression can be analyzed. However, in order to increase the number of genes that can be analyzed, it was necessary to repeat fluorescence measurement and chemiluminescence measurement in proportion to the number of measured genes.
  • the measurement takes into account the presence of multiple mRNA variants (variants) read from a single locus, and the various processes that occur during mRNA maturation and protein translation. Although the number of substances to be measured increases as necessary, here, in order to simplify the discussion, it refers to quantifying mature mRNA.
  • the mRNA corresponding to the gene expressed in one cell may be distributed from about several molecules to tens of thousands of molecules.
  • a single-cell-derived cDNA library is efficiently constructed (80% or more) on the surface of the beads described in Non-Patent Document 1, and the gene expression of multiple genes is accurately quantified by repeatedly measuring this.
  • the number of repetitions is limited to about 10-20, there is a problem that the number of measurable genes is also limited to 10-20.
  • the number of cells that can be measured simultaneously is about 100 cells or less, and the cost of the necessary reagents is also very expensive. Therefore, it is considered that it is an industrially important technique to measure only the number of genes that need to be measured for a large number of cells.
  • the cells are isolated and introduced into individual reaction wells, and reagents for cell disruption, reverse transcription, and PCR amplification are added to these reaction wells. Must be dispensed into. Therefore, in order to automate the analysis, a robot for dispensing is required, and the analysis apparatus becomes large and expensive.
  • the reaction channel in order to eliminate dispensing by robots, when extracting mRNA from cells using microfluidics and amplifying nucleic acids, the reaction channel must be arranged in a row, so the number of parallel lines is increased. Since the chip size increases in proportion, there is a problem that the size of the microfluidic device increases and becomes expensive.
  • the cells to be analyzed are introduced into the leftmost reaction tank, and the cells are crushed.
  • move the sample solution to the reaction tank on the right side perform the reverse transcription reaction, and further move the sample to the right to perform the PCR reaction.
  • Execute. Finally, collect the processed sample from the far right.
  • the solution needs to move in the substrate surface, which increases the footprint of the unit structure.
  • the arrangement of the flow path system for introducing the sample or reagent into the unit structure or the flow path system necessary for discharging the sample after processing consumes the chip area, the number of arrangements of the unit structure is increased. There is a problem that the chip area increases and the cost of the chip increases.
  • Patent Document 1 discloses a method using a cDNA library sheet in order to realize gene expression analysis of a large number of cells at a low cost at a time. Although it is possible to measure many cells at once, it was necessary to repeatedly measure fluorescence using a cDNA library in order to increase the number of genes that can be analyzed. Therefore, there was a limit to the number of genes analyzed.
  • the device and apparatus configuration are as follows.
  • the cell-derived sample flows in a direction perpendicular to the planar device surface, and the unit structure is arranged in a chip surface, thereby reducing the area where the unit structure is tightened on the chip.
  • the sample solution can be collected in a tag array that can be found by analyzing the sample in which position the unit structure is processed even if the sample processed by the unit structure arranged on the plane is mixed and recovered. (Tag molecule) is introduced in the course of sample processing. This eliminates the need for a sample collection mechanism for each unit structure.
  • the present invention includes the following inventions.
  • nucleic acid extraction device according to (1), wherein the nucleic acid trapping section includes beads to which DNA for nucleic acid trap is fixed.
  • nucleic acid extraction device wherein the nucleic acid trapping portion includes a porous membrane in which DNA for nucleic acid trap is fixed in a pore.
  • nucleic acid extraction device according to (2) or (3), wherein a part of the DNA for the nucleic acid trap includes a sequence for specifying a position on the chip.
  • nucleic acid extraction device according to (2) or (3), wherein a part of DNA for nucleic acid trap includes a different sequence for each trapped nucleic acid molecule.
  • nucleic acid extraction device further comprising means for introducing an enzyme for reverse transcription of RNA trapped in the nucleic acid trapping section.
  • nucleic acid extraction device according to any one of (1) to (8), wherein a nucleic acid trapping portion is provided immediately below the cell trapping portion.
  • nucleic acid extraction device according to any one of (1) to (8), wherein a nucleic acid trapping portion is provided in a region other than immediately below the cell trapping portion.
  • a nucleic acid processing apparatus comprising the nucleic acid extraction device according to any one of (1) to (10) and means for introducing a reagent for constructing a cDNA library.
  • a nucleic acid treatment comprising the nucleic acid extraction device according to any one of (1) to (10), a reagent for constructing a cDNA library, and a means for introducing a reagent for nucleic acid amplification apparatus.
  • nucleic acid extraction device according to any one of (1) to (10), and a microscope unit for observing cells trapped in the cell trapping unit with a differential interference microscope, a phase contrast microscope, a Raman microscope, or a coherent Raman microscope
  • a nucleic acid processing apparatus comprising:
  • a method for extracting nucleic acid from a cell using a nucleic acid extraction device comprising a cell trapping unit and a nucleic acid trapping unit disposed below the cell trapping unit, Contacting a cell with the cell trapping portion and trapping each one cell in the cell trapping portion; Flowing a nucleic acid extract for extracting nucleic acid from cells through a flow path passing through the cell trapping section from top to bottom; Immobilizing the extracted nucleic acid in the nucleic acid trapping portion; Discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section through a flow path, In the nucleic acid extraction device, the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction.
  • FIG. 1 (a) is a cross-sectional view perpendicular to the surface of the planar device
  • FIG. 1 (b) is a cross-sectional view taken along the alternate long and short dash line in FIG.
  • a cell trapping unit 2 for fixing cells 1 introduced into the device one by one in FIG. 1, the cell trapping unit has holes for fixing cells one by one
  • a nucleic acid for extracting nucleic acid from the cells The extraction liquid passes through the cell trapping section and flows from top to bottom, and is connected to the cell trapping section via the flow path and is arranged below the cell trapping section to fix the extracted nucleic acid.
  • a nucleic acid trapping section and a flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section.
  • the cell trapping section, the two flow paths, and the nucleic acid A nucleic acid extraction device characterized in that trapping portions form a pair in the vertical direction and a plurality of pairs are arranged in the plane direction is a basic structure of the present invention. This structure is configured on and in the flat substrate 6, and an upper reaction region 7 for introducing cells and a lower reaction region 8 for discharging processed nucleic acids are added as necessary.
  • the dotted arrow 9 is an example of the movement trajectory of the cell, and the arrow 10 indicates the movement direction of the nucleic acid extracted from the cell and the processed sample.
  • Non-Patent Document 2 a cell trap portion, a reverse transcription portion (RT) chamber, and a PCR portion (qPCR chamber) are arranged in the plane.
  • a larger area than that required for the cell trap must be provided for the two types of reaction vessels, and a valve and a flow path for controlling the liquid flow must also be provided in the plane. Therefore, a device for arranging a unit structure for processing one cell (in the structure of Non-Patent Document 2, a structure related to one cell processing including a cell trap part, a reverse transcription part, a PCR part, and a flow path system).
  • the upper area will become larger. Since the device cost is roughly proportional to the area of the device, increasing the number of cells that can be processed at the same time increases the device cost.
  • a nucleic acid trapping unit is provided immediately below the cell trapping unit, a cDNA library is constructed from the extracted nucleic acid in the nucleic acid trapping unit, and subsequent processing is performed on this library.
  • a plurality of reactions can be executed in one reaction region.
  • the nucleic acid trapping part preferably has a structure in which a large number of beads are packed or a porous structure.
  • the constructed cDNA is used as a template and nucleic acid amplification by PCR or transcription reaction is performed on the device, the sample is collected and quantified by sequencing using a next-generation sequencer. By making it differ depending on the trapped position, it becomes possible to identify which cell-derived gene expression level is the sequencing for the collected sample solution.
  • a reagent necessary for the nucleic acid trapping unit 4 is supplied from the upper reaction region 7 to cause a PCR reaction in the nucleic acid trapping unit, and a PCR amplification product is recovered from the lower reaction region 8.
  • nucleic acid trapping portion is not disposed but is disposed in the periphery.
  • FIG. 2 (a) is a cross-sectional view perpendicular to the surface of the planar device
  • FIG. 2 (b) is a cross-sectional view taken along the alternate long and short dash line in FIG.
  • the parts 21, 22, and 23 in the device use an optically transparent material.
  • a transmission microscope, differential interference microscope, phase contrast microscope, coherent standing Stokes Raman microscope (CARS microscope), or the like can be used.
  • sample and reagent are flowed in the direction of arrow 10 for nucleic acid extraction and reverse transcription.
  • the process of sample processing is the same as the case without microscopic observation.
  • the present embodiment relates to a nucleic acid extraction device and a sample processing apparatus in which a nucleic acid trapping unit is configured by packing a large number of DNAs (DNA probes) for nucleic acid traps immobilized on beads.
  • the basic structure of the unit structure of the nucleic acid extraction device of this example is the same as that shown in FIG. However, in this example, not only nucleic acid is extracted from cells and mRNA is captured, but a cDNA library is constructed, and this is used as a template, with a known sequence at the end that can be sequenced, and a sufficient amount of nucleic acid amplification product.
  • the device configuration is such that can be obtained.
  • FIG. 3 (a) shows a cross-sectional view of a unit structure for processing one cell in the nucleic acid extraction device corresponding to this example.
  • Figures 3 and 4 (b) to (f) show nucleic acid extraction and nucleic acid (mRNA) capture (b), cDNA synthesis (c), nucleic acid from each cell after capture of cells possible with this device.
  • a conceptual diagram of the steps of synthesis (d), (e) and PCR amplification (f) of the 2nd strand into which known terminal sequences necessary for amplification (PCR) and sequencing were introduced is shown.
  • FIG. 5 shows an overall configuration diagram of the nucleic acid extraction device.
  • FIG. 5 (a) is an overall view of a cross section of the nucleic acid extraction device corresponding to FIG.
  • FIG. 5 (b) is a cross-sectional view taken along the line AA ′ of FIG.
  • FIG. 5C is a cross-sectional view corresponding to the B-B ′ cross section of FIG.
  • FIG. 3 (a) in order to fix the cell 1 suspended in the solution to a specific position (cell trapping portion (opening)) 2 on the device, the upper reaction region 7 to the lower reaction region Allow the solution to flow through the cell trapping section towards 8.
  • the cells move along the flow of the solution and reach the cell trapping portion. Since the opening diameter of the cell trapping portion is smaller than the cell diameter, the cells are fixed here. Since the trapped cells act as plugs for the solution flow, the flow moves to a cell trapping section that has not yet captured the cells. Therefore, the remaining cells move to the part where the cells are not yet captured and are captured.
  • the solution that flows in the upper reaction region 7 is replaced with a nucleic acid extract such as Lysis buffer to destroy the cells (for example, a mixture of a surface active agent such as Tween 20 and a protease). Is done.
  • a nucleic acid extract such as Lysis buffer to destroy the cells
  • an electric field is applied in the direction of 11, and the nucleic acid (mRNA) in the cell is moved to the nucleic acid trapping unit 4 by electrophoresis.
  • the nucleic acid trapping unit 4 is a region between the channel 3 between the cell trapping unit 2 and the channel 5 between the lower reaction region 8, and a bead 12 on which a DNA probe 31 for trapping nucleic acid is fixed. Is the packed region.
  • FIG. 3 (b) shows an enlarged view of the surface of the bead 12 on which the DNA probe 31 is fixed.
  • An electric field 11 is applied so that the mRNA 32 extracted from the cells is captured on the beads in the nucleic acid trapping portion immediately below the captured cells.
  • the DNA probe 31 fixed to the beads includes a sequence that differs depending on the position of the nucleic acid trapping portion, that is, a cell recognition sequence.
  • the 3 'end of the DNA probe 31 has a poly T sequence, and the mRNA is captured by hybridizing with the poly A sequence at the 3' end of the mRNA.
  • Nucleic acid trapping sections arranged on the two-dimensional array shown in 4 of FIGS. 1 (b) and 5 (c) are packed with beads on which DNA probes 31 having different cell recognition sequences are immobilized. Yes.
  • the DNA probe 31 for capturing mRNA has a slightly more complicated sequence structure. As shown in FIG. 3 (b), a 30-base PCR amplification is performed from the 5 ′ end as the DNA probe 31.
  • Common sequence Forward direction
  • tag sequence for cell recognition of 5 bases tag sequence for molecular recognition consisting of a random sequence of 15 bases
  • dT oligo sequence of 18 bases + a VN sequence of 2 bases.
  • the common sequence for PCR amplification into the DNA probe 31, it can be used as a common primer in the subsequent PCR amplification step.
  • the oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of mRNA32 and is used to capture mRNA32 (FIG. 3 (b)).
  • a poly-T sequence was used as a part of the supplemental DNA probe 31 to analyze mRNA, but in order to perform microRNA or genomic analysis, a random sequence is complementary to the sequence to be analyzed instead of the poly-T sequence. Needless to say, a part of such a sequence may be used.
  • the first strand cDNA strand 33 is synthesized using the mRNA 32 captured by the DNA probe 31 on the beads as a template.
  • the voids of the beads packed with a solution containing reverse transcriptase and a synthetic substrate are filled, and the temperature is slowly raised to 50 ° C. and a complementary strand synthesis reaction is performed for about 50 minutes (FIG. 3 (c)).
  • RNase is flowed through the region packed with beads to decompose and remove mRNA32.
  • a solution containing an alkali modifier and a washing solution are passed through the voids of the beads to remove the residue and decomposition products.
  • a cDNA library array as shown in FIG. 5 (c) is constructed on the beads packed in the nucleic acid trapping portion by the process so far, reflecting the positions of individual cells captured by the cell trapping portion.
  • Lysis buffer is flowed from the lower reaction region 8 toward the upper reaction region 7 in order to remove cell debris remaining in the cell trapping portion.
  • multiple ( ⁇ 100) target gene-specific sequence primers 41 with a common sequence for PCR amplification (Reverse) added are annealed to the 1st cDNA strand (Fig. 4 (d)), and the 2nd cDNA strand is obtained by complementary strand extension reaction. 42 is synthesized (FIG. 4 (e)). That is, 2nd cDNA strand synthesis is performed under multiplex conditions.
  • a double-stranded cDNA chain having a common amplification sequence (Forward / Reverse) at both ends and containing a cell recognition tag, a molecular recognition tag, and a gene-specific sequence is synthesized.
  • PCR amplification was performed using a common sequence for amplification (Forward / Reverse) to prepare a PCR product 43 derived from a plurality of types of genes (FIG. 4 (f)). Even if an amplification bias occurs between genes or molecules in this step, high-accuracy quantitative data can be obtained because the amplification bias can be corrected using the molecular recognition tag after acquiring next-generation sequencer data. I can do it.
  • the number of mRNA per cell is approximately 10 6 , and 1.1 ⁇ 10 5 magnetic beads 12 are packed in the nucleic acid trap portion for capturing the mRNA.
  • Streptavidin is immobilized on the surface of the magnetic beads, and is modified to the surface of the magnetic beads via streptavidin by modifying biotin at the 5 ′ end of the DNA probe 31.
  • the nucleic acid trapping part, the cell trapping part, and the flow path connecting them packed with magnetic beads were produced as a substrate 6 made of PDMS (polydimethylsiloxane) using a semiconductor process.
  • PDMS polydimethylsiloxane
  • the cell trapping portion through holes having a diameter of 10 ⁇ m are arranged in an array at intervals of 125 ⁇ m.
  • the size of the substrate side is a square 13 mm, the cell trapping portion is disposed 10 4 therein.
  • the diameter of the through hole is as wide as 50 ⁇ m, and magnetic beads are packed in this part.
  • a pore array sheet (porous membrane) 35 was placed under the substrate 6 in which the through holes were arranged in an array.
  • the pore diameter of the pore array sheet is smaller than 1 ⁇ m which is the diameter of the magnetic beads.
  • the ink jet printer head is individually filled, and 2 nL of beads each having a different arrangement fixed are individually filled into the nucleic acid trapping unit 4.
  • the inner wall of the pore is processed into a hydrophilic surface and absorbs water but can hold the beads in the nucleic acid trapping portion.
  • pore array sheets such as monolithic sheets made of porous glass, capillary plates obtained by bundling capillaries and slicing, nylon membranes, and gel thin films.
  • a fine pore array sheet was used. Although such a sheet can be made by anodization, a sheet having a pore diameter of 20 nm to 200 nm and a diameter of 25 mm is commercially available. From this, it cut out into a square of 13 mm on a side and used it.
  • the pores formed in the sheet are the flow paths 5 that connect the nucleic acid trapping portion and the lower reaction region.
  • the PDMS substrate and the pore array sheet were bonded by plasma bonding.
  • a substrate made of a resin (polycarbonate, cyclic polyolefin, polypropylene) produced by nanoimprint technology or injection molding, a commercially available nylon mesh, or a track etch membrane may be used.
  • Adhesion with the pore array sheet can be performed by thermal adhesion.
  • this reaction layer may be integrally processed using a semiconductor process.
  • a magnetic bead solution (7 ⁇ 10 9 pieces / mL) with a diameter of 1 ⁇ m to which a DNA probe modified with a 5 ′ biotin group is immobilized is injected into the nucleic acid trapping unit 4 by 2 nL for each region by the same technique as an inkjet printer To do.
  • DNA probes having different cell recognition tag sequences (1024 types) for each region are discharged.
  • the magnetic bead solution is discharged through the channel 5, leaving only the beads.
  • the method for immobilizing mRNA capture DNA probes with different cell recognition tag sequences is to mix magnetic beads and DNA probe solution in separate reaction tubes and use Tris buffer (pH 7.4) containing 1.5M NaCl. Mix and incubate with rotation for 10 minutes.
  • the Lysis buffer is introduced from the upper inlet 305, the PBS buffer is discharged from the channels 306 and 307, and the upper reaction region 7 is replaced with the Lysis buffer.
  • the upper and lower sides of the nucleic acid extraction device are sandwiched between a transparent upper substrate 301 and a lower substrate 302.
  • Transparent (ITO) electrodes are formed inside these substrates by sputtering, an electric field is applied, and nucleic acids are moved to a nucleic acid trapping portion directly under the cells by electrophoresis.
  • the reason for making the electrode transparent is that the cells can be observed with an optical microscope.
  • the ITO transparent electrode used this time has a transmission characteristic of 40% or more in the wavelength range of 400 to 900 nm.
  • the distance between the upper electrode 301 and the lower electrode 302 is 2 mm, and the upper reaction region 7 and the lower reaction region 8 are completely filled with the Tris buffer.
  • the upper electrode 301 is used as the cathode (GND)
  • the lower electrode 302 is used as the anode
  • + 5V is applied for 2 minutes using the power supply 311.
  • mRNA is almost trapped in the oligo (dT) part of the DNA probe fixed to the beads. However, some mRNAs are not trapped by the secondary structure and move to the lower reaction region 8 below the beads.
  • To trap the mRNA completely with the DNA probe raise the temperature of the solution to 70 ° C, wait 5 minutes, and then reverse the polarity of the voltage applied to the lower electrode 302 every minute at -0.1 ° C / sec. It was cooled to 4 ° C. (at first, ⁇ 5 V was applied for 1 minute, and then repeatedly applied from +5 V to ⁇ 5 V 10 times per minute).
  • a cDNA immobilized on the surface of many beads for each cell was obtained as a library.
  • This is a so-called 1-cell cDNA library array, which is fundamentally different from the averaged cDNA library obtained from many cells so far.
  • the expression level for each gene is quantitatively measured for various genes from the cDNA library array thus obtained. Since there are 10,000 pores per cell, the average number of cDNA per pore is 100. For one type of cDNA, when the number of cDNA copies per cell is 10,000 or less, the average is 1 or less per bead.
  • PCR amplification step was performed (FIG. 4 (f)).
  • a heat block with heater (aluminum alloy or copper alloy) 309 and a temperature controller 310 may be added. This amplifies the target portion of 20 target genes, but the PCR product size is almost uniform at 200 ⁇ 8 bases. Collect the PCR amplification product solution accumulated in the solution.
  • PCR Purification Kit QIAGEN
  • emPCR amplification or bridge amplification After applying this solution to emPCR amplification or bridge amplification, the solution is applied to a next-generation sequencer of each company (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454) and analyzed.
  • FIG. 6 schematically shows a state where data sequenced in the same sequence except for the molecular recognition region is obtained (relevant portions of the obtained sequencing data are schematically shown).
  • 601, 602, 603, 604, and 605 are the same sequence including the molecular recognition tag sequence that is a random sequence, and show the cases where 1, 7, 4, 2, and 2 reads are obtained, respectively. Yes.
  • These sequences are all one molecule at the time when the 2nd strand is synthesized in FIG. 4 (e), and the number of molecules increases at the same time as the number of molecules increases by subsequent PCR amplification.
  • the same lead of the molecular recognition tag may be regarded as the same molecule, and all are regarded as one molecule.
  • the unevenness of the number of molecules for each sequence due to PCR amplification in the process after the synthesis of the second strand and adsorption to the inside of the pore array sheet when the solution is taken out is eliminated by the above-mentioned identification.
  • 1, 7, 4, 2, and 2 are apparent counts (the same sequence except for the molecular recognition tag).
  • the number of molecules in each cell is recounted as 1 count, for a total of 5 counts (1, 7, 4, 2, and 2 each corresponding to 1 count). That is, it is estimated that there were 5 molecules corresponding to sequences other than the molecular tag before amplification.
  • reads having different sequences other than the molecular recognition tag are also obtained as a sequencing result. At this time, by counting the number of reads in which the molecular recognition tags are different and the other sequences are the same, it is possible to execute the count for the sequence to be known. It can be presumed that the original sample contains mRNA with the number of molecules proportional to this count.
  • the prepared sheet can be used repeatedly, and for gene groups that need to know the expression level, a gene-specific sequence primer Mix solution with a common sequence primer (Reverse) for PCR amplification added is prepared. Similarly, synthesis of the 2nd cDNA strand, PCR amplification, and emPCR may be performed and analyzed by a next-generation sequencer. That is, by repeatedly using a cDNA library, it is possible to perform highly accurate expression distribution measurement for a necessary type of gene.
  • Example 2 beads were used to construct a cDNA library from a group of cells arranged in an array while retaining information on which cells derived mRNA contained in individual cells.
  • a nucleic acid extraction device having a nucleic acid trapping portion
  • a pore array sheet having a DNA probe immobilized thereon is used as the nucleic acid trapping portion.
  • T7 promoter is used instead of PCR amplification for nucleic acid amplification after constructing the cDNA library.
  • FIGS. 1-10 The structure of the nucleic acid extraction device and the extraction / treatment method using it in this example are shown in FIGS.
  • Fig. 7 (a) shows a sectional view of the unit structure of the nucleic acid extraction device.
  • the cells 1 are captured by the cell trapping unit 2 by flowing a buffer solution containing cells from the upper reaction region 7 to the lower reaction region 8 so as to penetrate the device.
  • the cell trapping unit 2 is in a cell array device 6 made of PDMS.
  • the diameter of the cell trapping part is 16 ⁇ m, which is slightly larger than the cell diameter. By setting the diameter, two or more cells are prevented from being trapped in the cell trapping portion.
  • the pore array sheet 71 that is a nucleic acid trapping portion has a large number of pores 72 penetrating the sheet, and a DNA probe is fixed to the inner wall of the pore 72.
  • a cell array device 6 made of PDMS for cell trap and a pore array sheet (porous membrane) 71 for nucleic acid trap are basic elements of the nucleic acid extraction device. These two elements are directly overlapped, and these elements play a role in the flow path connecting them.
  • the cell-containing buffer is replaced with Lysis buffer, and the cells are disrupted while an electric field is applied in a direction perpendicular to the device.
  • mRNA in the disrupted cell is captured by hybridization to the DNA probe 73 on the inner wall of the pore 72 immediately below the cell trap position.
  • the DNA probe 73 fixed inside the cell array sheet consists of a T7 promoter sequence from the 5 ′ end direction, a common sequence for emPCR amplification (Forward direction), a cell recognition tag sequence, a molecular recognition tag sequence, and an oligo (dT) sequence. Consists of.
  • the DNA probe when a transcription reaction from cDNA to cRNA by a transcription factor is performed in the nucleic acid amplification step, the DNA probe preferably further contains a promoter sequence of the transcription factor.
  • a promoter sequence As such a promoter sequence, T7 is used, but SP6, T3 and the like are also included. The activity of T7 RNA polymerase is used for nucleic acid amplification.
  • T7 promoter sequence is used, this sequence is recognized by T7 RNA polymerase, and a transcription (cRNA83 amplification) reaction is started from the downstream sequence.
  • Nucleic acid amplification using the promoter sequence of transcription factor is isothermal amplification, which not only eliminates the need for a temperature controller to add a temperature cycle, but also reduces the possibility of detachment of probe DNA immobilized on the device surface at high temperatures. can do.
  • a common sequence for PCR amplification it can be used as a common primer in the subsequent emPCR amplification step.
  • 4 5 1024 single cells by introducing a cell recognition tag (for example, 5 bases) into a DNA probe.
  • 4 7 1.6x10 4 molecules can be recognized by introducing a molecular recognition tag sequence (for example, 7 bases) into a DNA probe. Similar to the first embodiment, it is possible to recognize the origin. In other words, since the amplification bias between genes generated in the amplification process such as IVT / emPCR can be corrected, the amount of mRNA present in the sample can be quantified with high accuracy.
  • the oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of the mRNA and is used to capture the mRNA (FIG. 7 (a)).
  • pore array sheet As the pore array sheet, a commercially available product prepared by an anodic oxidation method is available. Here, a pore array sheet 71 having a pore diameter of 200 nm, a thickness of 60 ⁇ m, and a 13 mm square (cut from a 25 mm diameter sheet) is used. An example using this will be described.
  • the pores 72 formed in the pore array sheet 71 penetrate in the thickness direction of the pore array sheet 71, and the pores are completely independent.
  • the pore 72 also functions as the flow path 5.
  • the surface is hydrophilic, the protein adsorption to the surface is extremely small, and the enzymatic reaction proceeds efficiently.
  • the surface of the pore array sheet 71 is treated such as silane coupling to fix the DNA probe 73 to the pore surface. Since the DNA probe 73 is fixed to the surface at an average ratio of 30 to 100 nm 2 , 4 to 10 ⁇ 10 6 DNA probes are fixed to one hole.
  • the surface is coated with a surface coating agent in order to prevent surface adsorption. This surface coating may be performed simultaneously with the probe fixation.
  • the DNA probe density is such that mRNA passing through this space can be captured by the DNA probe with an efficiency of almost 100%.
  • the surface of the pores inside the pore array sheet is a surface that does not adsorb nucleic acids such as mRNA and PCR amplification primers, and proteins such as reverse transcriptase and polymerase at the same time that DNA probes are immobilized at high density.
  • a silane coupling agent for immobilizing a DNA probe and a silanized MPC polymer for preventing adsorption were simultaneously covalently immobilized on the pore surface at an appropriate ratio to increase the DNA We realized density fixation and stable adsorption inhibition of nucleic acids and proteins.
  • the alumina pore array sheet is first immersed in an ethanol solution for 3 minutes, then treated with UVO3 for 5 minutes, and washed with ultrapure water three times.
  • MPC 0.8 -MPTMSi 0.2 MPC: 2-Methacryloyloxyethyl phosphorylcholine / MPTMSi: 3-Methacryloxypropyl trimethoxysilane
  • GTMSi 3-Glycidoxypropyltrimethoxysilane Shin-Etsu Chemical
  • DNA probes containing different cell recognition tag sequences (1024 types) for each 25 ⁇ m ⁇ 25 ⁇ m region of 100 pL were discharged. Then, it was made to react at 25 degreeC in a humidification chamber for 2 hours.
  • mRNA 74 is captured by an 18-base poly-T sequence that is complementary to the poly-A sequence at the 3 ′ end of mRNA, as in the previous example.
  • the 1st cDNA strand 79 is synthesized to construct a cDNA library (FIG. 7 (b)).
  • anneal the multiple ( ⁇ 100 species) target gene-specific sequence primers 80 corresponding to the gene to be quantified to the 1st cDNA strand 79 (Fig. 8 (c)
  • synthesize the 2nd cDNA strand 81 by complementary strand elongation reaction. FIG. 8 (d)).
  • 2nd cDNA strand synthesis is performed under multiplex conditions.
  • a double-stranded cDNA having a common amplification sequence (Forward / Reverse) at both ends and containing a cell recognition tag, a molecular recognition tag, and a gene-specific sequence is synthesized.
  • 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ⁇ 5 bases 109 ⁇ 8 bases upstream from the poly A tail of the target gene were used for the gene-specific sequence of OAZ1), and this resulted in an amplification product size of approximately 200 bases in the subsequent IVT amplification step. It is for unifying.
  • FIG. 10 having the unit structure of the nucleic acid extraction device shown in FIG. 10B is a cross-sectional view taken along the line A-A ′ in FIG. 10A
  • FIG. 10C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ in FIG.
  • the process is the same as in Example 1 until the cell 1 is introduced from the cell inlet 308, the cell is trapped in the cell trapping part 2, and the 1st cDNA strand is synthesized.
  • sterilized water 340 ⁇ L and AmpliScribe 10 X Reaction Buffer (EPICENTRE) 100 ⁇ L and 100mMAdATP 90 ⁇ L and 100mM dCTP 90 ⁇ L and 100mM dGTP ⁇ 90 ⁇ L and 100mM dUTP 90 ⁇ L and 100mM DTT, and AmpliSzybe
  • the solution filling the upper reaction region 7 and the lower reaction region 8 was discharged from the outlets 306 and 307, and the solution containing reverse transcriptase was immediately injected from the inlet 305. Thereafter, the temperature of the solution was raised to 37 ° C. and maintained for 180 minutes to complete the reverse transcription reaction, and cRNA amplification was performed.
  • cRNA amplification product solution accumulated in the solution inside and outside the pores of the membrane.
  • PCR® Purification Kit QIAGEN
  • 50 ⁇ L of sterile water 50 ⁇ L of sterile water.
  • Example 3 It is possible to identify the individuality / state of a cell by a nucleic acid extraction device that realizes gene analysis for each cell. On the other hand, in non-invasive microscope observation, it is possible to measure cell morphology and chemical composition while keeping cells alive. However, it is extremely difficult to distinguish the state of a cell from information only from a microscopic image because the individuality / state of the cell is various and unstable.
  • a device and an apparatus configuration for combining identification of cell individuality by gene analysis for each cell and noninvasive imaging are shown. When microscopic observation is performed with cells captured using the device structure shown in FIG. 3 (a) or FIG.
  • FIG. 11 shows the structure and apparatus of the nucleic acid extraction device in this example.
  • FIG. 11B is a cross-sectional view taken along the line A-A ′ of FIG. 11A
  • FIG. 11C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ of FIG.
  • the shape of the substrate 6 made of PDMS was devised so that a nucleic acid trapping portion was not arranged just below the cells but directly below, and a peripheral ring-shaped region was provided and the magnetic beads 12 were packed in this portion.
  • nucleic acids such as mRNA are guided by the electrophoretic guide to the bead portion packed in a ring shape and captured by the DNA probe on the bead surface.
  • the following process is the same as that in Example 1.
  • the diameter of the cell trapping part prepared by PDMS was 16 ⁇ m
  • the diameter of the microscope window 1101 immediately below the cell trapping part was 25 ⁇ m
  • the height was 15 ⁇ m.
  • the pore array sheet 35 is also protected by a resist mask at the time of anodization so that pores are not formed in the portion corresponding to the portion immediately below the cell trapping portion. It is also possible to reduce the influence of scattering by the pore array sheet by making the thickness of the microscope window 1101 thicker than the depth of focus perpendicular to the device surface of the microscope optical system without performing anodization patterning. It is.
  • FIG. 12B is a cross-sectional view taken along the line A-A ′ in FIG. 12A
  • FIG. 12C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ in FIG.
  • 1201 is a nucleic acid trapping unit.
  • a DNA probe is immobilized on the beads and mRNA is captured.
  • Other device configurations and sample adjustment methods are the same as in the first embodiment.
  • the region packed with beads is used as a nucleic acid trapping portion, but in the same manner as in Example 2 in which the pore array sheet is used as a nucleic acid trapping portion, the portion where pores are formed is limited. Needless to say, it is possible to produce a configuration similar to that shown in 11 and 12 without using beads.
  • nucleic acid extraction device before performing detailed gene expression analysis by crushing cells for gene expression analysis, the shape and fluorescence staining of genes and proteins in high-resolution cells are alive. Quantitative or Raman imaging can be performed, and these imaging data can be associated with gene expression analysis data. A system configuration for realizing this will be described below.
  • cell samples placed on a planar device are measured with an optical microscope and gene expression analysis using the above device.
  • the minimum system configuration for measuring cell dynamics in detail by matching the results with individual cell data is shown.
  • 1200 represents a nucleic acid extraction device and a cell sample placed on the device.
  • 1201 is a flow system for performing mRNA extraction and nucleic acid amplification from cells represented by FIG.
  • this flow system by processing cell-derived mRNA, it has a certain length in order to determine the sequence with the next-generation (large-scale) DNA sequencer 1205, and the end is processed before nucleic acid processing.
  • An amplification product containing the tag sequence with information is obtained.
  • arrow 1211 indicates the movement of the amplification product.
  • the cells on the device are observed by the optical microscope 1203 in a form in which the positions of the cells on the device are specified in advance.
  • a thin arrow indicates movement of information.
  • the optical microscope 1203 includes a phase contrast microscope, a differential interference microscope, a fluorescence microscope, a laser-scanning confocal fluorescence microscope, a Raman microscope, a nonlinear Raman microscope (CARS microscope, SRS microscope, RIKE microscope), an IR microscope, and the like.
  • a phase contrast microscope a differential interference microscope
  • a fluorescence microscope a laser-scanning confocal fluorescence microscope
  • a Raman microscope a nonlinear Raman microscope (CARS microscope, SRS microscope, RIKE microscope)
  • an IR microscope and the like.
  • genetic information is concerned, there is little information obtained with these optical microscopes.
  • the measurement can be performed in a state where the cells are alive, the change with time of the cells can be measured, and the response of the cells to the stimulus can be measured in real time.
  • a device that stores position information on the device detailed information related to gene expression can be associated with information including temporal changes by a microscope.
  • an information system 1206 is provided in the system that integrates sequence information 1212 from the next generation (large-scale) DNA sequencer, optical microscope image information 1213, and position information 1214 corresponding to the tag sequence.
  • the minimum configuration of the system that integrates the cell measurement information described above is a system 1207 of a portion other than the next generation (large-scale) DNA sequencer 1205.
  • Fig. 13 shows a system configuration example when a fluorescence microscope is combined as an optical microscope.
  • 1203 is a fluorescence microscope.
  • GFP is expressed in a protein chamber (for example, p53) to be measured, or a fluorescent substance is introduced into a specific protein by immunostaining.
  • the amount of protein expressed in individual cells is correlated with the gene expression data obtained by processing the sample in a nucleic acid extraction device after cell disruption and quantifying it by DNA sequencing. Can take.
  • the nucleic acid is stained with DAPI and the cell nucleus is recognized, and the cell position is identified by a fluorescence microscope.
  • 1300 is a light source, here a mercury lamp.
  • 1301 is an excitation filter that determines the excitation wavelength
  • 1302 is a dichroic mirror
  • 1303 is an emission filter that selects a light reception wavelength.
  • 1301, 1302, and 1303 are selected by the control 1304, and only light from a specific phosphor is measured.
  • the fluorescence image of the cell is obtained by an objective lens 1305, an imaging lens 1306, and a CCD camera 1307.
  • 1308 is a control computer for controlling these and acquiring image data.
  • the control computer of the flow system is 1309, which controls the XY stage 1310 to move the microscope image.
  • the position coordinates on the microscopic image calculated using the position coordinates on the pore array sheet, the array data of the cell recognition tag and the XY stage position coordinates can be made to correspond on the control computer.
  • This control computer 1309 is a cell introduction control device 1311 that controls the introduction of cells into the flow cell system, a differentiation inducing agent that changes the state of the cell, a drug that wants to investigate the response of the cell, a lysate or sample for crushing the cell Reagent control device 1312 that controls the introduction of reagents for processing, cell culture conditions, temperature that controls the temperature cycle during PCR, CO 2 concentration control device 1313, unnecessary reagents and upper reagents used for cells, medium replacement, etc.
  • the discharge device 1314 and the lower reagent discharge device 1315 for discharging the prepared nucleic acid amplification product are appropriately controlled.
  • the finally obtained nucleic acid amplification product is passed to the next generation (large-scale) DNA sequencing system 1205 for sequence analysis.
  • emPCR and bridge amplifier for sequencing are assumed to be executed in this system.
  • the positional information of the image and the cell recognition tag sequence information collated on the control computer are sent to the integrated information system 1206 to associate the protein amount obtained from the fluorescence image with the gene expression amount.
  • the temporal change estimation of gene expression analysis data is executed with the same system. This makes it possible to measure the dynamics of the gene expression network.
  • This fluorescent microscope is used not only for intracellular measurement, but also for immunofluorescent staining of substances secreted from cells, such as captured cytokines, which are guided into the pore array sheet and captured by antibodies, and measure the amount thereof. Also good.
  • the gene expression level after disruption may be used in the same manner.
  • Fig. 14 shows an example in which a differential interference microscope is combined instead of a fluorescence microscope.
  • the differential interference microscope image only measures the shape without using a fluorescent reagent, but it is one of the measurement methods that has the least influence on cells when cells must be returned to the body, such as in regenerative medicine.
  • the measurement system is capable of performing detailed cell classification with little cell damage.
  • 1401 is a light source, here a halogen lamp.
  • 1402 is a polarizer
  • 1403 and 1404 are a Wollaston filter and a Wollaston prism, respectively.
  • 1405 is a condenser lens
  • 1406 is an objective lens.
  • FIG. 15 shows an example using a CARS microscope as an optical microscope.
  • the CARS microscope like the Raman microscope and IR microscope, can obtain a spectrum corresponding to the chemical species of the laser excitation part, and therefore can increase the amount of information on the cell state than the differential interference microscope.
  • CARS is a non-linear process, and has a merit that damage to cells is small because signal intensity is strong compared to Raman signal and sufficient signal can be obtained with relatively weak laser excitation intensity.
  • 1501 is a light source, here a pulse laser (microchip laser). This is split into two by a beam splitter 1502, and one is introduced into a nonlinear fiber (photonic crystal fiber) 1503 to generate Stokes light. The other light is used as it is as pump light and probe light, and is condensed on the sample (in the cell) by the water immersion objective lens 1504 to generate anti-Stokes light. Only the anti-Stokes light is transmitted through the high-pass filter 1505, and the coherent anti-Stokes Raman spectrum is acquired through the spectroscope 1506 by the CCD camera 1507 for the spectroscope.
  • a pulse laser microchip laser
  • the cell trapping portion is not composed of an opening having the same size as the cell, but a substance that chemically captures the cell surface, that is, a substance that chemically binds to the cell surface substance.
  • An example composed of fixed areas is shown.
  • FIG. 16 shows an example in which the cell trapping unit is changed corresponding to Example 1 (FIG. 1). Beads to which proteins such as antibodies that bind to the cell surface are immobilized are arranged in a partial region on the beads used for nucleic acid trapping (capture). The antibody on the bead can add a function of capturing a specific type of cell.
  • a group of antibodies called CD (cluster of differentiation) antibodies includes an antibody group corresponding to the type of cell membrane protein centering on leukocytes.
  • This antibody can be biotinylated, immobilized with streptavidin on the beads, and captured by using the inkjet printer technology on the cell trapping part 2 in Fig. 16 to capture cells with a specific CD classification antigen. It becomes.
  • the CD antibody is not directly immobilized on the beads, but may be immobilized on the beads via a biotin-modified secondary antibody.
  • antibodies other than CD antibodies may be immobilized on beads, or molecules that bind to receptors on cells may be immobilized.
  • An example of such a molecule is fibronectin. Fibronectin is known to bind to integrins on cells. By fixing fibronectin on the beads, it becomes possible to capture adherent cells.
  • substances that chemically bind to substances on the cell surface include extracellular matrices such as collagen, laminin, and elastin.
  • FIG. 17 shows a configuration example of the nucleic acid extraction device.
  • Cell capturing antibody 1702 is fixed to cell trapping section 2.
  • the immobilization method is by immobilizing biotinylated antibody and streptavidin in this region. Only cells having the antigen 1703 corresponding to the antibody can be captured.
  • Example 7 an example corresponding to Example 2 (FIG. 7) is shown.
  • a cell trapping part capable of capturing cells having the antigen 1802 corresponding to the antibody is formed. is doing.
  • about a few tens of pL of biotinylated antibody was injected using an inkjet printer head. Since the entire device surface is coated with streptavidin by the method shown in Example 3, the antibody is immobilized only in a specific region.
  • Example 5 In Examples 1 to 4, the second strand formation step (steps shown in FIGS. 2 (d)-(e) and 8 (c)-(d)) and the PCR amplification step (FIG. 2 (f) and FIG. 8). (e)) also shows an example performed in the apparatus as shown in FIG. 5, FIG. 10, FIG. 11 or FIG. However, the entire nucleic acid extraction device 1901 or the nucleic acid extraction device divided into multiple devices can be placed in a resin tube 1902 (commonly used 0.2 mL or 1.5 mL capacity tubes) or 96 or 384-well plates. Insertion as shown in FIG. 19 may be performed by mixing the reagent 1903 necessary for 2nd strand synthesis and PCR amplification into this tube. With this configuration, not only can the user freely change the conditions for these processes, but the cell recognition tag and molecular recognition tag can be changed from the 2nd strand synthesis in the tube. Can be inserted at the end opposite to the position shown in Example 1-4.
  • quantification, sequencing, and molecular identification of biomolecules can be performed on a large number of cultured cells, a large number of immune cells, (in blood) cancer cells, and the like. It is possible to measure whether or not a certain number exists in the living body. This makes it possible to measure early diagnosis of cancer and the heterogeneity of iPS cells.

Abstract

To express a number of genes in a number of cells, it was necessary to separate the cells, extract the genes therefrom, then amplify nucleic acids, and analyze the expression of the genes by sequence analysis. However, separation of cells seriously injures the cells and an expensive system is required therefor. According to the present invention, the expression of genes in individual cells is highly accurately analyzed by: arranging a pair of structures including a cell-trapping section and a nucleic acid-trapping section in a vertical direction to extract individual genes in cells; synthesizing cDNAs in the nucleic acid-trapping section; amplifying nucleic acids; and then analyzing the sequences thereof with a next generation sequencer.

Description

遺伝子定量・配列解析用２次元セルアレイデバイスおよび装置Two-dimensional cell array device and apparatus for gene quantification and sequence analysis
　本発明は遺伝子発現解析、細胞機能解析、生体組織の解析方法および病気の診断、創薬などに関するものである。詳しくは1細胞レベルでのmRNA解析方法に関するものである。 The present invention relates to gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, drug discovery, and the like. Specifically, it relates to an mRNA analysis method at the level of one cell.
　遺伝子発現解析では細胞群の中からmRNAを取り出し、相補鎖であるcDNAを作製してこれをPCRなどでコピー数を増幅し、DNAプローブアレー（DNAチップ）を用いてターゲットを対応するプローブ位置に捕獲して蛍光検出する方法が用いられている。しかし、PCR増幅やDNAチップを用いる方法は定量分析精度が低く、精度の高い遺伝子発現プロフィール分析方法が望まれていた。ヒトゲノム解析の終了に伴い遺伝子発現を定量的に調べる要求は強まりつつあるが、最近では1つの細胞からmRNAを抽出して定量分析を行う方法が望まれている。定量性の良い分析方法として定量PCRがあるが、これはターゲットと同じDNA配列を持つ標準試料を用意し、同じ条件下でPCR増幅して増幅の進行具合を蛍光プローブでモニターして比較することで定量分析を行う方法である。ターゲットが1細胞の場合にはもともとあったmRNAの数が少なく定量分析しにくい。また、複数の遺伝子発現の定量分析を行うには試料を分割してそれぞれ独立に定量分析を行うため、対象となる遺伝子の数が多く、発現量が少ない遺伝子が含まれる場合には試料分割により測定できない場合もある。 In gene expression analysis, mRNA is extracted from a group of cells, a complementary strand cDNA is prepared, its copy number is amplified by PCR, etc., and the target is placed at the corresponding probe position using a DNA probe array (DNA chip). A method of capturing and detecting fluorescence is used. However, methods using PCR amplification and DNA chips have low quantitative analysis accuracy, and a highly accurate gene expression profile analysis method has been desired. With the completion of human genome analysis, there is an increasing demand for quantitatively examining gene expression. Recently, a method of extracting mRNA from one cell and performing quantitative analysis is desired. Quantitative PCR is an analytical method with good quantitativeness. This involves preparing a standard sample with the same DNA sequence as the target, performing PCR amplification under the same conditions, and monitoring the progress of amplification with a fluorescent probe for comparison. This is a method for quantitative analysis. When the target is a single cell, the number of mRNA originally present is small and difficult to quantitatively analyze. In addition, for quantitative analysis of multiple gene expressions, the sample is divided and quantitative analysis is performed independently, so if there are many genes of interest and genes with low expression levels are included, In some cases, it cannot be measured.
　このような状況下で発明者らは全てのmRNAをcDNAに変え、ビーズ上に保持したcDNAライブラリ（全てのcDNAを含んだcDNA集合体）を作製して定量分析に利用する方法を考案した。そこではcDNAライブラリを繰り返し利用することで試料分割による微量発現遺伝子の計測ミスを取り除き、1細胞中に含まれる複数遺伝子の発現量を正確に計測できることを示した（非特許文献１）。 Under these circumstances, the inventors have devised a method for converting all mRNAs into cDNAs and preparing a cDNA library (cDNA aggregate containing all cDNAs) held on beads for use in quantitative analysis. There, it was shown that the expression level of a plurality of genes contained in one cell can be accurately measured by eliminating a measurement error of a small amount of expressed gene by dividing a sample by repeatedly using a cDNA library (Non-patent Document 1).
　上記方法は、手作業によるサンプルを含む溶液への反応試薬の混合、精製等のプロセスをすべて手作業で行っている。そのため、分注精度と溶媒の蒸発の問題から反応溶液のボリュームは数百ナノリットル～マイクロリットルオーダー以上に制限される。それゆえ、単一の細胞を解析する場合においても、適切な濃度の試薬を使わなければならないため、反応試薬量（モル数）が反応ボリュームに比例して増加し、それに比例して試薬コストが増加する。多数の細胞を計測しなければ統計的に有意なデータが得られない場合、その試薬コストは非常に大きな額となる。それゆえ、分注精度と蒸発の問題がない構造で反応ボリュームを低減する方法が求められている。 In the above method, all processes such as mixing and purification of reaction reagents into a solution containing a sample by hand are performed manually. Therefore, the volume of the reaction solution is limited to the order of several hundred nanoliters to microliters or more due to problems of dispensing accuracy and solvent evaporation. Therefore, even when analyzing a single cell, since an appropriate concentration of reagent must be used, the amount of the reaction reagent (number of moles) increases in proportion to the reaction volume, and the reagent cost increases proportionally. To increase. If statistically significant data cannot be obtained unless a large number of cells are measured, the reagent cost is very large. Therefore, there is a need for a method for reducing the reaction volume with a structure that does not have dispensing accuracy and evaporation problems.
　この問題を解決するために従来は、マイクロフルイディクスと呼ばれる小さな流路を組み合わせたデバイスを用いる方法が採用されてきた。単一細胞解析のためにマイクロフルイディクスを応用した例は非特許文献２に記載されている。非特許文献２中の図1にデバイスの構造図が記載されている。ここに記載されているチップは300個の細胞を同時に処理できるように一つの単位構造が300個配置されている。この単位構造がチップ上に3×50個配置されており、単位構造は横長で、この長手方向にサンプル溶液が流れ、流れながら、逐次反応をする構造となっている。反応ボリュームについては、逆転写反応時は10nLでPCR反応時は50nLであり、ボリュームが低減されている。 In order to solve this problem, conventionally, a method using a device combined with small flow channels called microfluidics has been adopted. An example of applying microfluidics for single cell analysis is described in Non-Patent Document 2. FIG. 1 in Non-Patent Document 2 describes a device structure diagram. The chip described here has 300 unit structures arranged so that 300 cells can be processed simultaneously. 3 × 50 unit structures are arranged on the chip. The unit structure is horizontally long, and the sample solution flows in the longitudinal direction and sequentially reacts while flowing. The reaction volume is 10 nL during the reverse transcription reaction and 50 nL during the PCR reaction, and the volume is reduced.
　さらに、たくさんの細胞を同時に処理するためには、多数の細胞を一度に低コストで遺伝子発現解析する必要がある。これを実現するために、ビーズの代わりに多孔質メンブレンなどを利用してcDNAライブラリを構成する方法が特許文献１に開示されている。この方法では、遺伝子発現の２次元分布を得て多数の細胞中の遺伝子発現解析を実現できるデバイスを用いている。このデバイスを用いて単一細胞中の遺伝子を発現解析するときには、細胞を単離する必要がなく、生体組織の切片中の細胞から、直接mRNAを取り出し、遺伝子発現解析することもできる。しかし、解析可能な遺伝子の数を増やすために繰り返し、計測遺伝子数に比例して蛍光計測や化学発光計測を行う必要があった。 Furthermore, in order to process many cells at the same time, it is necessary to perform gene expression analysis at a low cost on many cells at once. In order to realize this, Patent Document 1 discloses a method of constructing a cDNA library using a porous membrane or the like instead of beads. This method uses a device that can obtain a two-dimensional distribution of gene expression and realize gene expression analysis in a large number of cells. When analyzing the expression of a gene in a single cell using this device, it is not necessary to isolate the cell, and mRNA can be directly extracted from a cell in a section of a living tissue and the gene expression can be analyzed. However, in order to increase the number of genes that can be analyzed, it was necessary to repeat fluorescence measurement and chemiluminescence measurement in proportion to the number of measured genes.
特開2009-276883号公報JP 2009-276883
　再生医療、遺伝子を用いた診断、あるいは生命現象の基本的な理解には組織の平均としての遺伝子の発現量ばかりでなく、組織を構成する１つ１つの細胞の中味を定量的に分析することが重要視され始めている。これには細胞を１つずつ取り出して詳細に解析することとあわせて、統計的に有意なデータを取得するために多数の細胞について、細胞中の生体物質を定量することが望まれる。特に、種々遺伝子の発現量を定量的にモニターすることが望まれている。このとき、定量対象となる生体物質は細胞中のmRNAとなる。ここでは簡単のため、遺伝子とmRNAは１対１に対応しているものと考え、遺伝子発現定量することとmRNAを定量することは同じ意味であるとする。一般には、「遺伝子発現を測定する」という方が広い意味である。この場合には一つの遺伝子座から読みだされたmRNAのバリエーション（バリアント）が複数存在することや、mRNAの成熟過程や蛋白へ翻訳される過程で様々な制御を受けることも考慮された測定となり、測定対象物質が必要に応じて増えることとなるが、ここでは議論を簡略化するために成熟したmRNAを定量することを指すことにする。 For basic understanding of regenerative medicine, diagnosis using genes, or life phenomena, not only the expression level of genes as an average of tissues, but also quantitative analysis of the contents of each cell that constitutes tissues Is beginning to be emphasized. For this purpose, it is desired to quantify the biological material in a large number of cells in order to obtain statistically significant data in combination with taking out the cells one by one and analyzing them in detail. In particular, it is desired to quantitatively monitor the expression levels of various genes. At this time, the biological substance to be quantified becomes mRNA in the cell. Here, for simplicity, it is assumed that the gene and the mRNA have a one-to-one correspondence, and quantifying gene expression and quantifying mRNA have the same meaning. In general, “measuring gene expression” has a broader meaning. In this case, the measurement takes into account the presence of multiple mRNA variants (variants) read from a single locus, and the various processes that occur during mRNA maturation and protein translation. Although the number of substances to be measured increases as necessary, here, in order to simplify the discussion, it refers to quantifying mature mRNA.
　１細胞中に発現している遺伝子に対応するmRNAは、数分子数程度から数万分子まで分布している可能性がある。分子数の少ない遺伝子に対応するmRNAを精度良く定量するために、効率よく、定量可能な形に変換する必要がある。そのために、非特許文献１に記載のビーズ上表面に１細胞由来のcDNAライブラリを効率よく（80%以上）構築し、これを繰り返し計測することによって、精度良く複数遺伝子の遺伝子発現を定量することは可能となったが、この繰り返し回数は10～20回程度に制限されてしまうため、計測可能遺伝子数も10～20に制限されるという問題があった。同時に計測可能な細胞数は100細胞程度以下であり、必要な試薬の費用も非常に高価である。そのため、多数の細胞について、多数であるが、計測が必要な遺伝子数だけを計測するようにできることは、産業上重要な技術であると考えられる。 The mRNA corresponding to the gene expressed in one cell may be distributed from about several molecules to tens of thousands of molecules. In order to accurately quantify mRNA corresponding to a gene with a small number of molecules, it is necessary to efficiently convert it into a form that can be quantified. For this purpose, a single-cell-derived cDNA library is efficiently constructed (80% or more) on the surface of the beads described in Non-Patent Document 1, and the gene expression of multiple genes is accurately quantified by repeatedly measuring this. However, since the number of repetitions is limited to about 10-20, there is a problem that the number of measurable genes is also limited to 10-20. The number of cells that can be measured simultaneously is about 100 cells or less, and the cost of the necessary reagents is also very expensive. Therefore, it is considered that it is an industrially important technique to measure only the number of genes that need to be measured for a large number of cells.
　また、単一細胞中の遺伝子発現解析を実行するためには、一度細胞を単離して、個別の反応ウェルに導入し、細胞破砕や逆転写さらにはPCR増幅のための試薬をこれらの反応ウェルに分注しなければならない。そのため、解析の自動化を行うためには、分注のためのロボットが必要となり、解析装置が大型化し高価になってしまう。 In order to perform gene expression analysis in single cells, the cells are isolated and introduced into individual reaction wells, and reagents for cell disruption, reverse transcription, and PCR amplification are added to these reaction wells. Must be dispensed into. Therefore, in order to automate the analysis, a robot for dispensing is required, and the analysis apparatus becomes large and expensive.
　さらに、ロボットによる分注を排除するために、マイクロフルイディクスを用いて細胞からmRNAを抽出し、核酸増幅する場合においては、反応液流路を列状に配列しなければならないため、並列数に比例してチップサイズが大きくなるため、マイクロフルィディックデバイスのサイズが大型化して、高価になってしまうという問題点があった。 Furthermore, in order to eliminate dispensing by robots, when extracting mRNA from cells using microfluidics and amplifying nucleic acids, the reaction channel must be arranged in a row, so the number of parallel lines is increased. Since the chip size increases in proportion, there is a problem that the size of the microfluidic device increases and becomes expensive.
　実際には、非特許文献２の図1の左下にあるように、解析対象である細胞を一番左の反応槽に導入し、細胞を破砕する。その次に、右側の反応槽にサンプル溶液を移動させ、逆転写反応を実行し、さらに右に移動してPCR反応を行うというように一方方向に平面内でサンプルを移動させながら、サンプル処理を実行する。最後に、一番右から処理済みのサンプルを回収する。マイクロフルイディクスを用いる場合には溶液は基板面内を移動する必要があるため、単位構造のフットプリントが大きくなってしまう。また、単位構造へのサンプルや試薬を導入する流路系の配置や処理後のサンプルの排出に必要な流路系の配置のためにチップ面積を消費するため単位構造の配列数を増やそうとするとチップ面積が増大しチップのコストが上がってしまうという問題があった。 Actually, as shown in the lower left of FIG. 1 of Non-Patent Document 2, the cells to be analyzed are introduced into the leftmost reaction tank, and the cells are crushed. Next, move the sample solution to the reaction tank on the right side, perform the reverse transcription reaction, and further move the sample to the right to perform the PCR reaction. Execute. Finally, collect the processed sample from the far right. In the case of using microfluidics, the solution needs to move in the substrate surface, which increases the footprint of the unit structure. In addition, if the arrangement of the flow path system for introducing the sample or reagent into the unit structure or the flow path system necessary for discharging the sample after processing consumes the chip area, the number of arrangements of the unit structure is increased. There is a problem that the chip area increases and the cost of the chip increases.
　この非特許文献２では、細胞中のmRNAのうちのどの程度の割合がサンプルとして処理され、計測・定量できるようにされているかについては不明であるが、単一細胞解析中のmRNAなどの生化学物質を定量するためには、効率よく、ターゲット分子（特にmRNA）を計測できるようにすることは本質的な課題である。例えば、一細胞中に10分子程度以下しか存在しないmRNAも計測しなければならないが、計測できるように処理されたサンプルの分子数が少なくなると本質的な計測誤差（ポアソン分布に従うサンプリング誤差）が生じてしまう。 In this non-patent document 2, it is unclear as to what percentage of mRNA in a cell is processed as a sample so that it can be measured and quantified. In order to quantify chemical substances, it is an essential task to enable efficient measurement of target molecules (especially mRNA). For example, mRNA that has less than 10 molecules in one cell must be measured, but if the number of molecules in the sample processed so that it can be measured decreases, an inherent measurement error (sampling error according to the Poisson distribution) occurs. End up.
　また、多数の細胞を一度に低コストで遺伝子発現解析を実現するために、cDNAライブラリシートを用いる方法が特許文献１に開示されている。多数の細胞の一括計測が可能であるが、解析可能な遺伝子の数を増やすために繰り返し、cDNAライブラリを用いて蛍光計測する必要があった。そのため、解析遺伝子数に制限があった。 Further, Patent Document 1 discloses a method using a cDNA library sheet in order to realize gene expression analysis of a large number of cells at a low cost at a time. Although it is possible to measure many cells at once, it was necessary to repeatedly measure fluorescence using a cDNA library in order to increase the number of genes that can be analyzed. Therefore, there was a limit to the number of genes analyzed.
　上記課題、すなわち、一度に計測できる細胞数を増やすとともに細胞中の分子（ここではmRNA）を効率よくサンプル処理し、安価であるような計測デバイスまたは装置を提供するために、本発明では、以下のようなデバイスおよび装置構成をとる。 In order to provide the above-mentioned problem, that is, to increase the number of cells that can be measured at one time and to efficiently sample molecules (here, mRNA) in the cells and to provide a measurement device or apparatus that is inexpensive, The device and apparatus configuration are as follows.
　細胞を所定の位置に固定し、定量対象分子であるmRNAを核酸から抽出し、逆転写してcDNAライブラリを構築するなどのサンプル処理を細胞ごとに実行できる単位構造を２次元平面状に配置する。細胞由来のサンプルが流れる方向は、平面状のデバイス面に垂直に対して、単位構造をチップ面状に配置することによって、単位構造がチップ上で締める面積の低減を図る。また、サンプル溶液の回収は平面上に配置された単位構造で処理済のサンプルを混合して回収しても、どの位置の単位構造で処理されたかがサンプルを解析することで判明するようなタグ配列（タグ分子）をサンプル処理の過程で導入する。これによって、単位構造ごとに個別にサンプル回収のための機構が不要となる。 ∙ Place unit structures in a two-dimensional plane so that sample processing can be performed for each cell, such as fixing cells in place, extracting mRNA, which is a molecule to be quantified, from nucleic acid, and reverse transcription to construct a cDNA library. The cell-derived sample flows in a direction perpendicular to the planar device surface, and the unit structure is arranged in a chip surface, thereby reducing the area where the unit structure is tightened on the chip. In addition, the sample solution can be collected in a tag array that can be found by analyzing the sample in which position the unit structure is processed even if the sample processed by the unit structure arranged on the plane is mixed and recovered. (Tag molecule) is introduced in the course of sample processing. This eliminates the need for a sample collection mechanism for each unit structure.
　すなわち、本発明は以下の発明を包含する。 That is, the present invention includes the following inventions.
(1)それぞれ１つの細胞を固定するための細胞トラッピング部と、
　前記細胞から核酸を抽出するための核酸抽出液が前記細胞トラッピング部を通過して上から下へ流れる流路と、
　前記流路を介して前記細胞トラッピング部と繋がり前記細胞トラッピング部より下方に配置され、抽出された核酸を固定する核酸トラッピング部と、
　核酸抽出後の溶液を前記核酸トラッピング部から前記細胞トラッピング部とは反対側に排出する流路とを備え、
　前記細胞トラッピング部と、前記２つの流路と、前記核酸トラッピング部が上下方向に対を形成し、この対が、平面方向に複数配置されている、ことを特徴とする核酸抽出デバイス。 (1) a cell trapping unit for fixing one cell each;
A flow path through which the nucleic acid extract for extracting nucleic acid from the cells flows from the top to the bottom through the cell trapping section;
A nucleic acid trapping section that is connected to the cell trapping section via the flow path and is arranged below the cell trapping section and fixes the extracted nucleic acid;
A flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping part to the opposite side of the cell trapping part,
A nucleic acid extraction device, wherein the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction.
(2)前記核酸トラッピング部が、核酸トラップのためのDNAが固定されたビーズを含むことを特徴とする(1)記載の核酸抽出デバイス。 (2) The nucleic acid extraction device according to (1), wherein the nucleic acid trapping section includes beads to which DNA for nucleic acid trap is fixed.
(3)前記核酸トラッピング部が、細孔に核酸トラップのためのDNAが固定された多孔質メンブレンを含むことを特徴とする(1)記載の核酸抽出デバイス。 (3) The nucleic acid extraction device according to (1), wherein the nucleic acid trapping portion includes a porous membrane in which DNA for nucleic acid trap is fixed in a pore.
(4)前記細胞トラッピング部に、細胞表面の物質に化学的に結合する物質が固定されていることを特徴とする(1)～(3)のいずれかに記載の核酸抽出デバイス。 (4) The nucleic acid extraction device according to any one of (1) to (3), wherein a substance that chemically binds to a cell surface substance is fixed to the cell trapping portion.
(5)前記核酸トラップのためのDNAの一部がチップ上の位置を特定するための配列を含むことを特徴とする(2)または(3)記載の核酸抽出デバイス。 (5) The nucleic acid extraction device according to (2) or (3), wherein a part of the DNA for the nucleic acid trap includes a sequence for specifying a position on the chip.
(6)前記核酸トラップのためのDNAの一部がトラップした核酸分子ごとに異なる配列を含むことを特徴とする(2)または(3)記載の核酸抽出デバイス。 (6) The nucleic acid extraction device according to (2) or (3), wherein a part of DNA for nucleic acid trap includes a different sequence for each trapped nucleic acid molecule.
(7)前記核酸トラッピング部にトラップしたRNAを逆転写するための酵素を導入する手段を有することを特徴とする(6)記載の核酸抽出デバイス。 (7) The nucleic acid extraction device according to (6), further comprising means for introducing an enzyme for reverse transcription of RNA trapped in the nucleic acid trapping section.
(8)前記細胞トラッピング部の直下が光学的に透明な材料で構成されていることを特徴とする(1)～(7)のいずれかに記載の核酸抽出デバイス。 (8) The nucleic acid extraction device according to any one of (1) to (7), wherein the cell trapping portion is made of an optically transparent material.
(9)前記細胞トラッピング部の直下に核酸トラッピング部を設けたことを特徴とする(1)～(8)のいずれかに記載の核酸抽出デバイス。 (9) The nucleic acid extraction device according to any one of (1) to (8), wherein a nucleic acid trapping portion is provided immediately below the cell trapping portion.
(10)前記細胞トラッピング部の直下以外の領域に核酸トラッピング部を設けたことを特徴とする(1)～(8)のいずれかに記載の核酸抽出デバイス。 (10) The nucleic acid extraction device according to any one of (1) to (8), wherein a nucleic acid trapping portion is provided in a region other than immediately below the cell trapping portion.
(11)(1)～(10)のいずれかに記載の核酸抽出デバイスと、cDNAライブラリを構築するための試薬を導入する手段とを有することを特徴とする核酸処理装置。 (11) A nucleic acid processing apparatus comprising the nucleic acid extraction device according to any one of (1) to (10) and means for introducing a reagent for constructing a cDNA library.
(12)(1)～(10)のいずれかに記載の核酸抽出デバイスと、cDNAライブラリを構築するための試薬と核酸増幅のための試薬を導入する手段とを有することを特徴とする核酸処理装置。 (12) A nucleic acid treatment comprising the nucleic acid extraction device according to any one of (1) to (10), a reagent for constructing a cDNA library, and a means for introducing a reagent for nucleic acid amplification apparatus.
(13)(1)～(10)のいずれかに記載の核酸抽出デバイスと、細胞トラッピング部にトラップした細胞を微分干渉顕微鏡、位相差顕微鏡、ラマン顕微鏡またはコヒーレントラマン顕微鏡で観察するための顕微鏡部とを有することを特徴とする核酸処理装置。 (13) The nucleic acid extraction device according to any one of (1) to (10), and a microscope unit for observing cells trapped in the cell trapping unit with a differential interference microscope, a phase contrast microscope, a Raman microscope, or a coherent Raman microscope A nucleic acid processing apparatus comprising:
(14)細胞トラッピング部と、前記細胞トラッピング部より下方に配置される核酸トラッピング部とを備えた核酸抽出デバイスにより、細胞から核酸を抽出する方法であって、
　前記細胞トラッピング部に細胞を接触させて、それぞれ１つの細胞を前記細胞トラッピング部にトラップする工程と、
　細胞から核酸を抽出するための核酸抽出液を、前記細胞トラッピング部を通過して上から下へ通じる流路を通して流す工程と、
　前記核酸トラッピング部に抽出された核酸を固定する工程と、
　核酸抽出後の溶液を、前記核酸トラッピング部から前記細胞トラッピング部とは反対側に流路を介して排出する工程とを含み、
　前記核酸抽出デバイスにおいて、前記細胞トラッピング部と、前記２つの流路と、前記核酸トラッピング部が上下方向に対を形成し、この対が、平面方向に複数配置されていることを特徴とする前記方法。 (14) A method for extracting nucleic acid from a cell using a nucleic acid extraction device comprising a cell trapping unit and a nucleic acid trapping unit disposed below the cell trapping unit,
Contacting a cell with the cell trapping portion and trapping each one cell in the cell trapping portion;
Flowing a nucleic acid extract for extracting nucleic acid from cells through a flow path passing through the cell trapping section from top to bottom;
Immobilizing the extracted nucleic acid in the nucleic acid trapping portion;
Discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section through a flow path,
In the nucleic acid extraction device, the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction. Method.
　単一細胞中の核酸などの生体分子を定量したり、配列決定したり、生体分子の種類を同定するデバイスにおいて、一度に計測できる細胞数を増やすとともに、細胞中の分子（例えばmRNA）を効率よくサンプル処理でき安価な計測デバイスまたは装置を提供することができる。 In devices that quantify and sequence biomolecules such as nucleic acids in a single cell, and identify the type of biomolecule, increase the number of cells that can be measured at one time and increase the efficiency of molecules in the cell (eg, mRNA) It is possible to provide an inexpensive measuring device or apparatus that can perform sample processing well.
本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの使用方法の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the usage method of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスで用いる核酸の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid used with the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの使用方法の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the usage method of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの使用方法の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the usage method of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸処理装置の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid processing apparatus of this invention. 本発明の核酸処理装置の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid processing apparatus of this invention. 本発明の核酸処理装置の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid processing apparatus of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. 本発明の核酸抽出デバイスの処理方法の一実施形態を示す概略図である。It is the schematic which shows one Embodiment of the processing method of the nucleic acid extraction device of this invention.
　本発明の核酸抽出デバイスの具体的な構造は、例えば図1に例示できる。図1(a)は面状デバイスの面に垂直方向の断面図で、図1(b)は、面に並行で上の図の一点鎖線部分の位置での断面図である。デバイス中に導入された細胞1を1つずつ固定する細胞トラッピング部2（図1では、細胞トラッピング部は細胞を1つずつ固定する穴を有する）と、前記細胞から核酸を抽出するための核酸抽出液が前記細胞トラッピング部を通過して上から下へ流れる流路3と、前記流路を介して前記細胞トラッピング部と繋がり前記細胞トラッピング部より下方に配置され、抽出された核酸を固定する核酸トラッピング部4と、核酸抽出後の溶液を前記核酸トラッピング部から前記細胞トラッピング部とは反対側に排出する流路5とを備え、前記細胞トラッピング部と、前記２つの流路と、前記核酸トラッピング部が上下方向に対を形成し、この対が、平面方向に複数配置されている、ことを特徴とする核酸抽出デバイスが本発明の基本となる構造である。この構造は平面基板6上および内部に構成され、細胞を導入するための上部反応領域7と処理済の核酸を排出するための下部反応領域8も必要に応じて追加する。点線の矢印9は細胞の移動の軌跡の例であり、矢印10は細胞から抽出された核酸および処理されたサンプルの移動方向を示す。 The specific structure of the nucleic acid extraction device of the present invention can be illustrated, for example, in FIG. FIG. 1 (a) is a cross-sectional view perpendicular to the surface of the planar device, and FIG. 1 (b) is a cross-sectional view taken along the alternate long and short dash line in FIG. A cell trapping unit 2 for fixing cells 1 introduced into the device one by one (in FIG. 1, the cell trapping unit has holes for fixing cells one by one), and a nucleic acid for extracting nucleic acid from the cells The extraction liquid passes through the cell trapping section and flows from top to bottom, and is connected to the cell trapping section via the flow path and is arranged below the cell trapping section to fix the extracted nucleic acid. A nucleic acid trapping section; and a flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section. The cell trapping section, the two flow paths, and the nucleic acid A nucleic acid extraction device characterized in that trapping portions form a pair in the vertical direction and a plurality of pairs are arranged in the plane direction is a basic structure of the present invention. This structure is configured on and in the flat substrate 6, and an upper reaction region 7 for introducing cells and a lower reaction region 8 for discharging processed nucleic acids are added as necessary. The dotted arrow 9 is an example of the movement trajectory of the cell, and the arrow 10 indicates the movement direction of the nucleic acid extracted from the cell and the processed sample.
　非特許文献２に記載されているデバイスでは細胞トラップ部（cell capture chamber）と逆転写部（RT chamber）とPCR部（qPCR chamber）が面内に配置されている。細胞トラップに必要な面積よりも大きな面積を別に２種類の反応槽向けに配置しなければならず、さらに、液流を制御するためのバルブや流路も面内に配置しなければならない。そのため、１つの細胞を処理するための単位構造（非特許文献２の構造では細胞トラップ部と逆転写部とPCR部と流路系を含む一つの細胞処理にかかわる構造）を配置するためのデバイス上の面積が大きくなってしまう。デバイスコストは大雑把にはデバイスの面積に比例することから、同時処理細胞数を増やすとデバイスコストの上昇を招いてしまう。 In the device described in Non-Patent Document 2, a cell trap portion, a reverse transcription portion (RT) chamber, and a PCR portion (qPCR chamber) are arranged in the plane. A larger area than that required for the cell trap must be provided for the two types of reaction vessels, and a valve and a flow path for controlling the liquid flow must also be provided in the plane. Therefore, a device for arranging a unit structure for processing one cell (in the structure of Non-Patent Document 2, a structure related to one cell processing including a cell trap part, a reverse transcription part, a PCR part, and a flow path system). The upper area will become larger. Since the device cost is roughly proportional to the area of the device, increasing the number of cells that can be processed at the same time increases the device cost.
　図1の実施形態では、この問題を解決するために核酸トラッッピング部を細胞トラッピング部の直下に設け、抽出した核酸から核酸トラッピング部でcDNAライブラリを構築し、その後の処理をこのライブラリに対して実行することで複数の反応を１つの反応領域で実行できるようにしている。 In the embodiment of FIG. 1, in order to solve this problem, a nucleic acid trapping unit is provided immediately below the cell trapping unit, a cDNA library is constructed from the extracted nucleic acid in the nucleic acid trapping unit, and subsequent processing is performed on this library. Thus, a plurality of reactions can be executed in one reaction region.
　核酸トラップの効率を上げるために、核酸トラッピング部は多数のビーズが詰め込まれた構造や多孔質構造が望ましい。このような単位容積中の反応場となる表面積を増やすことによって、小さな反応槽でも効率が高く、短時間での反応が可能となる。 In order to increase the efficiency of the nucleic acid trap, the nucleic acid trapping part preferably has a structure in which a large number of beads are packed or a porous structure. By increasing the surface area as a reaction field in such a unit volume, the efficiency is high even in a small reaction tank, and the reaction can be performed in a short time.
　さらに、構築されたcDNAを鋳型にして、PCRや転写反応などによる核酸増幅をデバイス上で実行後にサンプルを回収して、次世代シーケンサを用いたシーケンスによって定量するとき、構築されたcDNAの配列がトラップされた位置によって異なるようにすることで、回収されたサンプル溶液に対するシーケンシングがどの細胞由来の遺伝子発現量かを同定することが可能となる。 Furthermore, when the constructed cDNA is used as a template and nucleic acid amplification by PCR or transcription reaction is performed on the device, the sample is collected and quantified by sequencing using a next-generation sequencer. By making it differ depending on the trapped position, it becomes possible to identify which cell-derived gene expression level is the sequencing for the collected sample solution.
　これによって、単位構造を隔離したり、サンプルの移動を制御するためのバルブ機構や流路が不要となる。 This eliminates the need for a valve mechanism or flow path for isolating the unit structure and controlling the movement of the sample.
　図1の構成では、核酸トラッピング部4に必要な試薬を上部反応領域7から供給することによって核酸トラッピング部でPCR反応させ、PCR増幅産物は下部反応領域8から回収する。このとき、デバイス全体の温度がPCR反応に適切な温度サイクルとなるような機構をデバイスの外側に設けることが好ましい。 In the configuration of FIG. 1, a reagent necessary for the nucleic acid trapping unit 4 is supplied from the upper reaction region 7 to cause a PCR reaction in the nucleic acid trapping unit, and a PCR amplification product is recovered from the lower reaction region 8. At this time, it is preferable to provide a mechanism on the outside of the device so that the temperature of the entire device becomes a temperature cycle suitable for the PCR reaction.
　さらに望ましい構造として、細胞の状態を事前に非侵襲顕微鏡を用いて計測し、同一の細胞について遺伝子発現解析などで細胞中の生体物質を定量できるようにするために、細胞を固定した直下には核酸トラッピング部を配置せず、周辺に配置する構造を取ることが例示できる。 Furthermore, as a desirable structure, in order to measure the state of cells using a non-invasive microscope in advance and to quantify biological substances in the cells by gene expression analysis etc. for the same cells, directly under the cells fixed An example is a structure in which the nucleic acid trapping portion is not disposed but is disposed in the periphery.
　具体的には図2に示すような構造である。図2(a)は面状デバイスの面に垂直方向の断面図で、図2(b)は、面に並行で上の図の一点鎖線部分の位置での断面図である。細胞1から核酸を抽出する前に顕微鏡観察を行うためにデバイス中の21、22、23の部分は光学的に透明な材料を用いる。顕微鏡観察には透過型顕微鏡、微分干渉顕微鏡、位相差顕微鏡、コヒーレント安置ストークスラマン顕微鏡(CARS顕微鏡)等を用いることができる。 Specifically, the structure is as shown in FIG. FIG. 2 (a) is a cross-sectional view perpendicular to the surface of the planar device, and FIG. 2 (b) is a cross-sectional view taken along the alternate long and short dash line in FIG. In order to perform microscopic observation before extracting the nucleic acid from the cell 1, the parts 21, 22, and 23 in the device use an optically transparent material. For the microscopic observation, a transmission microscope, differential interference microscope, phase contrast microscope, coherent standing Stokes Raman microscope (CARS microscope), or the like can be used.
　顕微鏡観察終了後、核酸抽出および逆転写を行うために矢印10の方向にサンプルをおよび試薬を流す。サンプル処理の過程は顕微鏡観察をしない場合と同じである。 After completion of microscopic observation, the sample and reagent are flowed in the direction of arrow 10 for nucleic acid extraction and reverse transcription. The process of sample processing is the same as the case without microscopic observation.
（実施例1）
　本実施例は、ビーズ上に固定した核酸トラップのためのDNA（DNAプローブ）を多数パッキングすることによって核酸トラッピング部を構成した核酸抽出デバイスおよびサンプル処理装置に関する実施例である。 (Example 1)
The present embodiment relates to a nucleic acid extraction device and a sample processing apparatus in which a nucleic acid trapping unit is configured by packing a large number of DNAs (DNA probes) for nucleic acid traps immobilized on beads.
　本実施例の核酸抽出デバイスの単位構造の基本構成は図1に示したものと同じである。ただし、本実施例では、細胞から核酸を抽出し、mRNAを捕捉するだけでなく、cDNAライブラリを構築し、これを鋳型として、シーケンシング可能な末端に既知配列をもって、かつ十分量の核酸増幅産物が得られるようなデバイスの構成となっている。 The basic structure of the unit structure of the nucleic acid extraction device of this example is the same as that shown in FIG. However, in this example, not only nucleic acid is extracted from cells and mRNA is captured, but a cDNA library is constructed, and this is used as a template, with a known sequence at the end that can be sequenced, and a sufficient amount of nucleic acid amplification product. The device configuration is such that can be obtained.
　本実施例に対応する核酸抽出デバイスの中の一つの細胞を処理するための単位構造の断面図を図3(a)に記した。また、図3、図4の(b)～(f)に、このデバイスで可能な細胞の捕捉後の各細胞から核酸抽出および核酸(mRNA)捕捉(b)、cDNAの合成(c)、核酸増幅(PCR)およびシーケンスに必要な既知の末端配列を導入した2ndストランドの合成(d)、(e)およびPCR増幅(f)のステップの概念図を記した。また、図5に核酸抽出デバイスの全体の構成図を記した。図5(a)は、図1(a)、図3(a)に対応する核酸抽出デバイスの断面の全体図であり、図5(b)は図5(a)のA-A’断面であり、図1(b)に対応する図面である。また、図5(c)は図5(a)のB-B’断面に対応する断面図である。 FIG. 3 (a) shows a cross-sectional view of a unit structure for processing one cell in the nucleic acid extraction device corresponding to this example. Figures 3 and 4 (b) to (f) show nucleic acid extraction and nucleic acid (mRNA) capture (b), cDNA synthesis (c), nucleic acid from each cell after capture of cells possible with this device. A conceptual diagram of the steps of synthesis (d), (e) and PCR amplification (f) of the 2nd strand into which known terminal sequences necessary for amplification (PCR) and sequencing were introduced is shown. Further, FIG. 5 shows an overall configuration diagram of the nucleic acid extraction device. FIG. 5 (a) is an overall view of a cross section of the nucleic acid extraction device corresponding to FIG. 1 (a) and FIG. 3 (a), and FIG. 5 (b) is a cross-sectional view taken along the line AA ′ of FIG. There is a drawing corresponding to FIG. FIG. 5C is a cross-sectional view corresponding to the B-B ′ cross section of FIG.
　次に、核酸抽出デバイスの動作について、処理順に説明する。図3(a)において、溶液に懸濁された細胞1をデバイス上の特定の位置（細胞トラッピング部（開口部））2に固定されるようにするために、上部反応領域7から下部反応領域8に向かって細胞トラッピング部を溶液が通って流れるようにする。細胞は溶液の流れに乗って移動し、細胞トラッピング部まで到達し、細胞トラッピング部の開口径が細胞の直径よりも小さいため、ここに細胞が固定される。捕捉された細胞は溶液流に対して栓の役割を果たすので、流れはまだ細胞を捕捉していない細胞トラッピング部に移動する。そのため、残りの細胞はまだ細胞が捕捉されていない部分へと移動し捕捉される。目的の数だけ細胞1が捕捉されたら、上部反応領域7に流す溶液は細胞を破壊するためのLysis bufferなどの核酸抽出液（例えばTween20などの表面活性剤と蛋白分解酵素の混合液）に交換される。同時に11の方向に電界を印加し、細胞中の核酸(mRNA)を核酸トラッピング部4まで電気泳動によって移動させる。核酸トラッピング部4は、細胞トラッピング部2との間の流路3と下部反応領域8との間の流路5の間の領域で、核酸をトラップするためのDNAプローブ31が固定されたビーズ12がパッキングされた領域である。 Next, the operation of the nucleic acid extraction device will be described in the order of processing. In FIG. 3 (a), in order to fix the cell 1 suspended in the solution to a specific position (cell trapping portion (opening)) 2 on the device, the upper reaction region 7 to the lower reaction region Allow the solution to flow through the cell trapping section towards 8. The cells move along the flow of the solution and reach the cell trapping portion. Since the opening diameter of the cell trapping portion is smaller than the cell diameter, the cells are fixed here. Since the trapped cells act as plugs for the solution flow, the flow moves to a cell trapping section that has not yet captured the cells. Therefore, the remaining cells move to the part where the cells are not yet captured and are captured. When the desired number of cells 1 has been captured, the solution that flows in the upper reaction region 7 is replaced with a nucleic acid extract such as Lysis buffer to destroy the cells (for example, a mixture of a surface active agent such as Tween 20 and a protease). Is done. At the same time, an electric field is applied in the direction of 11, and the nucleic acid (mRNA) in the cell is moved to the nucleic acid trapping unit 4 by electrophoresis. The nucleic acid trapping unit 4 is a region between the channel 3 between the cell trapping unit 2 and the channel 5 between the lower reaction region 8, and a bead 12 on which a DNA probe 31 for trapping nucleic acid is fixed. Is the packed region.
　図3(b)にはDNAプローブ31が固定されたビーズ12表面の拡大図を示している。細胞から抽出されたmRNA32は捕捉された細胞の直下にある核酸トラッピング部にあるビーズ上に捕捉されるように電界11が印加されている。捕捉したmRNAが元々存在していた細胞の位置の情報を配列情報として保存するために、ビーズに固定されたDNAプローブ31は、核酸トラッピング部の位置によって異なる配列、すなわち、細胞認識配列を含む。このDNAプローブ31の3’末端はポリＴ配列を持っており、mRNAの3’末端のポリA配列とハイブリダイズすることによってmRNAを捕捉する。図1(b)および図5(c)の4で示された2次元アレイ上に配置された核酸トラッピング部にはそれぞれ異なる細胞認識配列をもったDNAプローブ31が固定されたビーズがパッキングされている。 FIG. 3 (b) shows an enlarged view of the surface of the bead 12 on which the DNA probe 31 is fixed. An electric field 11 is applied so that the mRNA 32 extracted from the cells is captured on the beads in the nucleic acid trapping portion immediately below the captured cells. In order to store the information on the position of the cell where the captured mRNA originally existed as sequence information, the DNA probe 31 fixed to the beads includes a sequence that differs depending on the position of the nucleic acid trapping portion, that is, a cell recognition sequence. The 3 'end of the DNA probe 31 has a poly T sequence, and the mRNA is captured by hybridizing with the poly A sequence at the 3' end of the mRNA. Nucleic acid trapping sections arranged on the two-dimensional array shown in 4 of FIGS. 1 (b) and 5 (c) are packed with beads on which DNA probes 31 having different cell recognition sequences are immobilized. Yes.
　このmRNA捕捉用DNAプローブ31は、本実施例ではもう少し複雑な配列構成となっており、図3(b)に示されているように、5’末端からDNAプローブ31として、30塩基のPCR増幅用共通配列（Forward方向）、5塩基の細胞認識用タグ配列、15塩基のランダム配列からなる分子認識用タグ配列、および18塩基のオリゴ(dT)配列＋2塩基のVN配列を含めた。PCR増幅用共通配列をDNAプローブ31へ導入する事で、後続のPCR増幅工程において共通プライマーとして利用する事ができる。また、細胞認識タグについては、例えば5塩基のランダム配列の場合、45=1024個の単一細胞を認識する事が可能となる。すなわち、1度に1024個の単一細胞からcDNAライブラリを調製する事ができ、上記に記したように最終的に得られる次世代シーケンサの配列データにおいて、どの細胞由来であるかを認識する事が可能となる。さらに、分子認識用タグ配列（例えば７塩基）をDNAプローブ31へ導入する事により、47=1.6x104分子を認識する事ができるため、次世代シーケンサで得られる増幅産物についてのDNA配列データから同じ細胞由来で同じ遺伝子の配列をもった増幅産物が、どの分子由来であるかを認識する事が可能となる。すわなち、増幅工程で生じた遺伝子間の増幅バイアスを修正する事ができるため、始めに試料中に存在していたmRNA量を高い精度で定量する事が可能となる。ただし、単一細胞中の同一遺伝子の発現量が分子認識タグ配列のバリエーションよりも多い場合には増幅バイアス補正の正確性が低下する。最も3’側に位置するオリゴ(dT)配列は、mRNA32の3’側に付加されているポリAテールとハイブリダイズし、mRNA32を捕捉するために利用される(図3(b))。 In this example, the DNA probe 31 for capturing mRNA has a slightly more complicated sequence structure. As shown in FIG. 3 (b), a 30-base PCR amplification is performed from the 5 ′ end as the DNA probe 31. Common sequence (Forward direction), tag sequence for cell recognition of 5 bases, tag sequence for molecular recognition consisting of a random sequence of 15 bases, and an oligo (dT) sequence of 18 bases + a VN sequence of 2 bases. By introducing the common sequence for PCR amplification into the DNA probe 31, it can be used as a common primer in the subsequent PCR amplification step. As for the cell recognition tag, for example, in the case of a random sequence of 5 bases, 4 5 = 1024 single cells can be recognized. That is, a cDNA library can be prepared from 1024 single cells at a time, and as described above, in the sequence data of the next-generation sequencer finally obtained, which cell is derived can be recognized. Is possible. Furthermore, 4 7 = 1.6x10 4 molecules can be recognized by introducing a molecular recognition tag sequence (for example, 7 bases) into the DNA probe 31, so that DNA sequence data for amplification products obtained with the next-generation sequencer Therefore, it is possible to recognize which molecule is derived from the amplification product having the same gene sequence derived from the same cell. In other words, since the amplification bias between genes generated in the amplification process can be corrected, the amount of mRNA present in the sample can be quantified with high accuracy. However, when the expression level of the same gene in a single cell is larger than the variation of the molecular recognition tag sequence, the accuracy of amplification bias correction is reduced. The oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of mRNA32 and is used to capture mRNA32 (FIG. 3 (b)).
　本実施例ではmRNAを解析するために補足用DNAプローブ31の一部にポリT配列を用いたが、microRNAやゲノム解析を行うためにポリT配列の代わりにランダム配列は解析したい配列と相補的な配列の一部を用いてもよいことは言うまでもない。 In this example, a poly-T sequence was used as a part of the supplemental DNA probe 31 to analyze mRNA, but in order to perform microRNA or genomic analysis, a random sequence is complementary to the sequence to be analyzed instead of the poly-T sequence. Needless to say, a part of such a sequence may be used.
　次にビーズ上のDNAプローブ31により捕捉したmRNA32を鋳型にして1st cDNA鎖33を合成する。本工程では逆転写酵素および合成基質を含む溶液でパックされたビーズの空隙部分を満たし、50℃にゆっくり昇温して50分ほど相補鎖合成反応を行う図3(c)。反応終了後RNaseを、ビーズがパッキングされた領域を通して流してmRNA32を分解除去する。次いでアルカリ変性剤を含む液および洗浄液をビーズの空隙部分に通して流し、残存物および分解物を除去する。ここまでのプロセスで核酸トラッピング部にパックされたビーズ上には、細胞トラッッピング部に捕捉された個々の細胞の位置を反映して図5(c)に示すようなcDNAライブラリアレイが構築される。 Next, the first strand cDNA strand 33 is synthesized using the mRNA 32 captured by the DNA probe 31 on the beads as a template. In this step, the voids of the beads packed with a solution containing reverse transcriptase and a synthetic substrate are filled, and the temperature is slowly raised to 50 ° C. and a complementary strand synthesis reaction is performed for about 50 minutes (FIG. 3 (c)). After completion of the reaction, RNase is flowed through the region packed with beads to decompose and remove mRNA32. Next, a solution containing an alkali modifier and a washing solution are passed through the voids of the beads to remove the residue and decomposition products. A cDNA library array as shown in FIG. 5 (c) is constructed on the beads packed in the nucleic acid trapping portion by the process so far, reflecting the positions of individual cells captured by the cell trapping portion.
　次に細胞トラッピング部に残存する細胞の破片を取り除くためにLysis bufferを下部反応領域8から上部反応領域7に向かって流す。引き続きPCR増幅用共通配列(Reverse)が付加された複数（~100種）のターゲット遺伝子特異的配列プライマー41を1st cDNA鎖へアニールさせ（図4(d)）、相補鎖伸長反応により2nd cDNA鎖42を合成させる(図4(e))。すなわちマルチプレックス条件で2nd cDNA鎖合成を行う。これにより、複数のターゲット遺伝子について、増幅用共通配列(Forward/Reverse)を両端に持ち、細胞認識タグ、分子認識タグ、および遺伝子特異的配列がその中に含まれる2本鎖cDNA鎖が合成される。また本実施例では、一例として、20種類(ATP5B、GAPDH、GUSB、HMBS、HPRT1、RPL4、RPLP1、RPS18、RPL13A、RPS20、ALDOA、B2M、EEF1G、SDHA、TBP、VIM、RPLP0、RPLP2、RPLP27およびOAZ1)の遺伝子特異的配列にターゲット遺伝子のポリAテールから109±8塩基上流部分の20±5塩基を用いたが、これは、後続のPCR増幅工程において、PCR産物サイズを約200塩基に統一するためである。PCR産物サイズを統一する事で、煩雑なサイズフラクション精製の工程(電気泳動→ゲルの切り出し→PCR産物の抽出・精製)を除去する事ができ、1分子からの並列増幅（エマルジョンPCRなど）へ直接利用できる効果を持つ。続いて、増幅用共通配列(Forward/Reverse)を用いてPCR増幅を行い、複数種の遺伝子由来のPCR産物43を調製した（図4(f)）。この工程において遺伝子間、ないし分子間で増幅バイアスが生じたとしても、次世代シーケンサデータ取得後に、分子認識タグを利用して増幅バイアスの補正を行う事ができるため、高精度な定量データを得る事が出来る。 Next, Lysis buffer is flowed from the lower reaction region 8 toward the upper reaction region 7 in order to remove cell debris remaining in the cell trapping portion. Next, multiple (~ 100) target gene-specific sequence primers 41 with a common sequence for PCR amplification (Reverse) added are annealed to the 1st cDNA strand (Fig. 4 (d)), and the 2nd cDNA strand is obtained by complementary strand extension reaction. 42 is synthesized (FIG. 4 (e)). That is, 2nd cDNA strand synthesis is performed under multiplex conditions. As a result, for a plurality of target genes, a double-stranded cDNA chain having a common amplification sequence (Forward / Reverse) at both ends and containing a cell recognition tag, a molecular recognition tag, and a gene-specific sequence is synthesized. The In this example, as an example, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27 and For the gene-specific sequence of OAZ1), 20 ± 5 bases upstream of 109 ± 8 bases from the poly A tail of the target gene were used, but this unified the PCR product size to about 200 bases in the subsequent PCR amplification process. It is to do. By unifying the PCR product size, complicated size fraction purification steps (electrophoresis-> gel excision-> PCR product extraction / purification) can be eliminated, and parallel amplification from one molecule (emulsion PCR, etc.) Has an effect that can be used directly. Subsequently, PCR amplification was performed using a common sequence for amplification (Forward / Reverse) to prepare a PCR product 43 derived from a plurality of types of genes (FIG. 4 (f)). Even if an amplification bias occurs between genes or molecules in this step, high-accuracy quantitative data can be obtained because the amplification bias can be corrected using the molecular recognition tag after acquiring next-generation sequencer data. I can do it.
　1細胞当りのmRNAの数はおおよそ106個であり、このmRNAを補足するための核酸トラップ部分には1.1×105個の磁性ビーズ12をパックしている。磁性ビーズ表面にはストレプトアビジンが固定さてており、DNAプローブ31の5’末端にビオチンを修飾することによってストレプトアビジンを介して磁性ビーズ表面に固定される。 The number of mRNA per cell is approximately 10 6 , and 1.1 × 10 5 magnetic beads 12 are packed in the nucleic acid trap portion for capturing the mRNA. Streptavidin is immobilized on the surface of the magnetic beads, and is modified to the surface of the magnetic beads via streptavidin by modifying biotin at the 5 ′ end of the DNA probe 31.
　ここまで、ビーズ表面で生成したcDNA(1st cDNA)にPCR増幅用の共通配列が付加されている数十種類の遺伝子特異的配列プライマーを用いて2nd cDNAを合成し、ここでは、PCR増幅する例を開示した。もちろんローリングサークル増幅(RCA)やNASBAやLAMP法など他の増幅法を用いても良い。 So far, we have synthesized 2nd cDNA using dozens of gene-specific sequence primers in which a common sequence for PCR amplification is added to the cDNA generated on the bead surface (1st cDNA). Disclosed. Of course, other amplification methods such as rolling circle amplification (RCA), NASBA, and LAMP may be used.
　次に、核酸抽出デバイスの作製方法について詳述する。磁性ビーズがパックされる核酸トラッピング部、細胞トラッッピング部およびこれらを結ぶ流路をPDMS(ポリジメチルシロキサン)製の基板6として半導体プロセスを用いて作製した。細胞トラッピング部は直径10μmの貫通孔を125μm間隔にアレイ状に配置する。基板の大きさは一辺が13mmの正方形であり、その中に細胞トラッピング部が104個配置されている。細胞トラッピング部の直下は貫通孔の直径が50μmに広くなっておりこの部分に磁性ビーズをパッキングする。この貫通孔がアレイ状に配置された基板6の下に細孔アレイシート(多孔質メンブレン)35を配置した。この細孔アレイシートの細孔の直径は磁性ビーズの直径である1μmよりも小さい。 Next, a method for producing a nucleic acid extraction device will be described in detail. The nucleic acid trapping part, the cell trapping part, and the flow path connecting them packed with magnetic beads were produced as a substrate 6 made of PDMS (polydimethylsiloxane) using a semiconductor process. In the cell trapping portion, through holes having a diameter of 10 μm are arranged in an array at intervals of 125 μm. The size of the substrate side is a square 13 mm, the cell trapping portion is disposed 10 4 therein. Directly under the cell trapping part, the diameter of the through hole is as wide as 50 μm, and magnetic beads are packed in this part. A pore array sheet (porous membrane) 35 was placed under the substrate 6 in which the through holes were arranged in an array. The pore diameter of the pore array sheet is smaller than 1 μm which is the diameter of the magnetic beads.
　その後、個別にインクジェットプリンタヘッドに充填し、異なる配列が固定されたビーズを個別に核酸トラッピング部4に2nLずつ充填する。 After that, the ink jet printer head is individually filled, and 2 nL of beads each having a different arrangement fixed are individually filled into the nucleic acid trapping unit 4.
　細孔の内壁は親水表面に加工されており、水を吸収するがビーズを核酸トラッピング部に保持することが可能である。細孔アレイシートとしては多孔質のガラスからなるモノリスシート、毛細管を束ねてスライスしたキャピラリープレート、ナイロンメンブレンあるいはゲル薄膜など種々のものを用いる可能性があるが、ここではアルミナを陽極酸化して得た細孔アレイシートを用いた。このようなシートは陽極酸化により自作することもできるが、孔径20nm～200nm、直径25mmのものが市販品として入手可能である。これから、一辺13mmの正方形に切り出して使用した。シートに形成された細孔は核酸トラッピング部と下部反応領域をつなげる流路5である。 The inner wall of the pore is processed into a hydrophilic surface and absorbs water but can hold the beads in the nucleic acid trapping portion. There are various types of pore array sheets, such as monolithic sheets made of porous glass, capillary plates obtained by bundling capillaries and slicing, nylon membranes, and gel thin films. A fine pore array sheet was used. Although such a sheet can be made by anodization, a sheet having a pore diameter of 20 nm to 200 nm and a diameter of 25 mm is commercially available. From this, it cut out into a square of 13 mm on a side and used it. The pores formed in the sheet are the flow paths 5 that connect the nucleic acid trapping portion and the lower reaction region.
　上記PDMS製基板と細孔アレイシートはプラズマ接着によって接着した。 The PDMS substrate and the pore array sheet were bonded by plasma bonding.
　ここで、PDMS製基板の代わりにナノインプリント技術や射出成型によって作製した樹脂（ポリカービネート、サイクリックポリオレフィン、ポリプロピレン）製の基板や、市販のナイロンメッシュやトラックエッチメンブレンを用いてもよい。細孔アレイシートとの接着は熱接着を用いることもできる。 Here, instead of the PDMS substrate, a substrate made of a resin (polycarbonate, cyclic polyolefin, polypropylene) produced by nanoimprint technology or injection molding, a commercially available nylon mesh, or a track etch membrane may be used. Adhesion with the pore array sheet can be performed by thermal adhesion.
　また、この反応層は半導体プロセスを用いて一体加工を行ってもよいことは言うまでもない。 Needless to say, this reaction layer may be integrally processed using a semiconductor process.
　次に、5’ビオチン基修飾されたDNAプローブが固定された直径１μmの磁性ビーズ溶液（７×109個/mL）を核酸トラッピング部4にインクジェットプリンタと同じ技術によって、2nLずつ領域ごとに注入する。このとき、領域ごとに異なる細胞認識タグ配列（1024種類）をもつDNAプローブを吐出する。磁性ビーズの溶液は流路5を通して排出され、ビーズだけが残る。なお、異なる細胞認識用タグ配列をもったmRNA捕捉用DNAプローブが固定する方法は、別々の反応チューブ中で磁性ビーズとDNAプローブ溶液を混和し1.5M NaClを含むTris バッファ(pH 7.4)中で混和し、10分回転させながら結合反応させる。 Next, a magnetic bead solution (7 × 10 9 pieces / mL) with a diameter of 1 μm to which a DNA probe modified with a 5 ′ biotin group is immobilized is injected into the nucleic acid trapping unit 4 by 2 nL for each region by the same technique as an inkjet printer To do. At this time, DNA probes having different cell recognition tag sequences (1024 types) for each region are discharged. The magnetic bead solution is discharged through the channel 5, leaving only the beads. The method for immobilizing mRNA capture DNA probes with different cell recognition tag sequences is to mix magnetic beads and DNA probe solution in separate reaction tubes and use Tris buffer (pH 7.4) containing 1.5M NaCl. Mix and incubate with rotation for 10 minutes.
　以下、これまで記述した、核酸抽出デバイスを作成し、次世代（大規模）シーケンサで遺伝子発現プロファイルを得るための装置システムを図5を用いて説明する。1000個程度以下の細胞を500μLの１×PBSで細胞を傷つけないように洗浄後、できる限りPBSが残らないように溶液を除去し、4℃に冷却された1×PBSバッファを50μL加えた。細胞は細胞インレット308から導入し、バッファを下部アウトレット307から排出することによって細胞を細胞トラップ部にアレイ状に配列させる。過剰な細胞は上部アウトレット306から排出する。次に上部インレット305からLysisバッファを導入し、PBSバッファを流路306および307から排出して、上部反応領域7をLysisバッファに置換する。核酸抽出デバイスの上下は透明な上基板301および下基板302で挟まれている。これらの基板内側に透明（ITO）電極をスパッタリングによって形成し、電界を印加して電気泳動によって核酸を細胞直下の核酸トラッピング部に移動させる。電極を透明にしている理由は、細胞を光学顕微鏡で観察できるようにするためであり、今回使用したITO透明電極は400-900nmの波長範囲で40％以上の透過特性を有する。 Hereinafter, an apparatus system for creating a nucleic acid extraction device described above and obtaining a gene expression profile with a next-generation (large-scale) sequencer will be described with reference to FIG. After washing about 1000 cells or less with 500 μL of 1 × PBS so as not to damage the cells, the solution was removed so as not to leave PBS as much as possible, and 50 μL of 1 × PBS buffer cooled to 4 ° C. was added. Cells are introduced from the cell inlet 308, and the buffer is discharged from the lower outlet 307, whereby the cells are arranged in an array in the cell trap portion. Excess cells drain from the upper outlet 306. Next, the Lysis buffer is introduced from the upper inlet 305, the PBS buffer is discharged from the channels 306 and 307, and the upper reaction region 7 is replaced with the Lysis buffer. The upper and lower sides of the nucleic acid extraction device are sandwiched between a transparent upper substrate 301 and a lower substrate 302. Transparent (ITO) electrodes are formed inside these substrates by sputtering, an electric field is applied, and nucleic acids are moved to a nucleic acid trapping portion directly under the cells by electrophoresis. The reason for making the electrode transparent is that the cells can be observed with an optical microscope. The ITO transparent electrode used this time has a transmission characteristic of 40% or more in the wavelength range of 400 to 900 nm.
　Lysis Solution 495uL（TaqMan MicroRNA Cell-to-CT Kit; Applied Biosystems Inc.）とDNase I 5uLをインレット305から注入する。溶液が、ゲル化したことを確認し、温度を20℃まで上げて、8分間反応させた後、Stopping Solution（DNaseを失活させる溶液） 50uLをゲルの上に添加し、5分間反応させ、4℃に冷却した。次に、分子量60万、0.03％のPEO(ポリエチレンオキシド)と分子量100万、0.03％のPVP(ポリビニルピロリドン)および、0.1%のTween 20を含む10mMのTris Buffer (pH 8.0)0.5mLを加えた。このとき上部電極301と下部電極302間の距離は2mmとし、上部反応領域7および下部反応領域8は前記Tris bufferで完全に満たされている。溶液温度を4℃に維持したまま、上部電極301を陰極(GND)、下部電極302を陽極として、電源311を用いて+5Vを２分間印加し、負電荷を持つmRNAを細胞内部から下部反応領域8の方向へ電気泳動する。電気泳動条件はDC電圧印加の代わりにオンレベル10Ｖ，オフレベル0Ｖ、周波数100ｋHz，デューティー50％のパルス電気泳動を用いてもよい。この過程でmRNAはビーズに固定されたDNAプローブのオリゴ(dT)部分にほとんどトラップされる。しかし、一部のmRNAは2次構造によってトラップされずにビーズ下部の下部反応領域8に移動してしまう。mRNAを完全にDNAプローブにてトラップするために、溶液の温度を70℃まで上げて5分待った後、1分毎に下部電極302に印加する電圧の極性を反転させながら-0.1℃/secで4℃まで冷却した（最初は-5Vを１分間印加し、その後+5V→-5Vで１分ずつ10回繰り返し印加）。次にインレット305から上記tris bufferを導入し、アウトレット306から排出することによって、上部反応領域7中の溶液を交換しながら、溶液の温度を35℃まで上げてアガロースゲルを溶解させて、必要のない細胞組織とアガロースを洗浄、除去した。さらに、0.1%Tween20を含む10mM Tris Buffer (pH=8.0) 585μLと10mM dNTP 40μLと5xRT Buffer (SuperScript III, Invitrogen社) 225μLと0.1M DTT 40μLとRNaseOUT (Invitrogen社) 40μLとSuperscript III(逆転写酵素, Invitrogen社) 40μLを混和し、上部反応領域7および下部反応領域8を満たしている溶液をアウトレット306および307から排出し、直ちに逆転写酵素を含む上記溶液をインレット305から注入した。その後、溶液の温度を50℃に上げて、50分間保つことによって逆転写反応を完了させ、mRNAと相補的配列を持つ1st cDNAを合成した。 Injection of 495uL of Lysis Solution 495uL (TaqMan MicroRNA Cell-to-CT Kit; Applied Biosystems Inc.) and DNase I 5uL from the inlet 305. After confirming that the solution has gelled, raise the temperature to 20 ° C and react for 8 minutes. Then add 50uL of Stopping Solution (solution that deactivates DNase) over the gel and react for 5 minutes. Cooled to 4 ° C. Next, 0.5 mL of 10 mM Tris buffer (pH 8.0) containing 600,000 0.03% PEO (polyethylene oxide), 1 million molecular weight, 0.03% PVP (polyvinylpyrrolidone) and 0.1% Tween 20 was added. . At this time, the distance between the upper electrode 301 and the lower electrode 302 is 2 mm, and the upper reaction region 7 and the lower reaction region 8 are completely filled with the Tris buffer. While maintaining the solution temperature at 4 ° C, the upper electrode 301 is used as the cathode (GND), the lower electrode 302 is used as the anode, and + 5V is applied for 2 minutes using the power supply 311. Electrophoresis in the direction of region 8. As the electrophoresis conditions, pulse electrophoresis with an on level of 10 V, an off level of 0 V, a frequency of 100 kHz, and a duty of 50% may be used instead of applying a DC voltage. During this process, mRNA is almost trapped in the oligo (dT) part of the DNA probe fixed to the beads. However, some mRNAs are not trapped by the secondary structure and move to the lower reaction region 8 below the beads. To trap the mRNA completely with the DNA probe, raise the temperature of the solution to 70 ° C, wait 5 minutes, and then reverse the polarity of the voltage applied to the lower electrode 302 every minute at -0.1 ° C / sec. It was cooled to 4 ° C. (at first, −5 V was applied for 1 minute, and then repeatedly applied from +5 V to −5 V 10 times per minute). Next, by introducing the above tris buffer from the inlet 305 and discharging from the outlet 306, while exchanging the solution in the upper reaction region 7, the temperature of the solution is raised to 35 ° C. to dissolve the agarose gel, No cell tissue and agarose were washed and removed. In addition, 10 mM Tris Buffer (pH = 8.0) containing 0.1% Tween20, 585 μL, 10 mM dNTP, 40 μL, and 5 x RT Buffer (SuperScript III, Invitrogen) 225 μL, 0.1 M DTT 40 μL, RNaseOUT (Invitrogen) 40 μL, and Superscript III (reverse transcriptase) , (Invitrogen)) 40 μL was mixed, the solution filling the upper reaction region 7 and the lower reaction region 8 was discharged from the outlets 306 and 307, and the solution containing reverse transcriptase was immediately injected from the inlet 305. Thereafter, the temperature of the solution was raised to 50 ° C. and kept for 50 minutes to complete the reverse transcription reaction, and 1st cDNA having a sequence complementary to mRNA was synthesized.
　細胞ごとに多くのビーズ表面に固定されたcDNAをライブラリとして得た。これはいわば１細胞cDNAライブラリアレイというべきものであり、これまでの多くの細胞から得られる平均化されたcDNAライブラリとは根本的に違うものである。 A cDNA immobilized on the surface of many beads for each cell was obtained as a library. This is a so-called 1-cell cDNA library array, which is fundamentally different from the averaged cDNA library obtained from many cells so far.
　このように得られたcDNAライブラリアレイから種々遺伝子について遺伝子ごとの発現量を定量的に計測する。１つの細胞当たり１万個の細孔があるので、１つの細孔当たりのcDNAの個数は平均で100個である。１種類のcDNAについて、１細胞当たりのcDNAコピー数が１万個以下の場合には、１つのビーズ当たり平均として１個以下となる。 The expression level for each gene is quantitatively measured for various genes from the cDNA library array thus obtained. Since there are 10,000 pores per cell, the average number of cDNA per pore is 100. For one type of cDNA, when the number of cDNA copies per cell is 10,000 or less, the average is 1 or less per bead.
　1st cDNA鎖を合成した後、85℃にて1.5分保ち逆転写酵素を失活させ、4℃に冷却後、RNaseおよび0.1%Tween20を含む10mM Tris Buffer (pH = 8.0) 10mLをインレット305から注入しアウトレット306、307から排出することによって、RNAを分解し、同量のアルカリ変性剤を同様に流して残存物および分解物を除去・洗浄した。続いて、滅菌水690μLと10 x Ex Taq Buffer (TaKaRa Bio社) 100μLと2.5mM dNTP Mix 100μLと各10μMのPCR増幅用共通配列(Reverse)が付加された20種の遺伝子特異的配列プライマー Mix100μLとEx Taq Hot start version (TaKaRa Bio社)10μLを混和し、デバイス中を満たしている溶液をアウトレット306および307から排出し、直ちに逆転写酵素を含む上記溶液をインレット305から注入した。その後、95℃3分間→44℃2分間→72℃6分間の反応を行い、1st cDNA鎖を鋳型としてプライマーの遺伝子特異的配列をアニールさせた後（図4(d)）、相補鎖伸長反応を行い、2nd cDNA鎖を合成させた（図4(e)）。 After synthesizing the 1st cDNA strand, incubate the reverse transcriptase for 1.5 minutes at 85 ° C, cool to 4 ° C, and inject 10 mL of 10 mM Tris Buffer (pH = 8.0) 含 む 10 mL containing RNase and 0.1% Tween20 from the inlet 305. Then, the RNA was decomposed by discharging from the outlets 306 and 307, and the same amount of alkali denaturant was flowed in the same manner to remove and wash the residue and degradation products. Subsequently, 690 μL of sterilized water, 10 x Ex Taq Buffer (TaKaRa Bio) 100 μL, 2.5 mM dNTP Mix 100 μL, and 20 gene-specific sequence primers Mix 100 μL each with 10 μM PCR amplification consensus sequence (Reverse) added. 10 μL of Ex Taq Hot version (TaKaRa Bio) was mixed, the solution filling the device was discharged from outlets 306 and 307, and the above solution containing reverse transcriptase was immediately injected from inlet 305. Then, 95 ° C for 3 minutes → 44 ° C for 2 minutes → 72 ° C for 6 minutes, annealing the gene-specific sequence of the primer using the 1st cDNA strand as a template (Figure 4 (d)), and then complementary strand extension reaction To synthesize a 2nd cDNA strand (FIG. 4 (e)).
　続いて、滅菌水495μLと10 x High Fidelity PCR Buffer (Invitrogen) 100μLと2.5 mM dNTP mix 100μLと50mM MgSO4 40μLと10μMのPCR増幅用共通配列プライマー (Forward) 100μLと10μMのPCR増幅用共通配列プライマー(Reverse) 100μLとPlatinum Taq Polymerase High Fidelity (Invitrogen社) 15μLを混和し、上部反応領域7および下部反応領域8を満たしている溶液をアウトレット306および307から排出し、直ちに上記溶液をインレット305から注入した。その後、溶液を30秒間94℃に保ち、94℃30秒間→55℃30秒間→68℃30秒間の3段階工程を40サイクル繰り返し、最後に68℃3分間保った後、4℃に冷却してPCR増幅工程を行った(図4(f))。このような温度サイクルを実現するために、ヒーター付きヒートブロック（アルミ合金または銅合金）309と温度コントローラ310を追加してもよい。これにより、20種のターゲット遺伝子の目的部分が増幅されるが、いずれもPCR産物サイズは200±8塩基とほぼ均一である。溶液中に蓄積されたPCR増幅産物溶液を回収する。この溶液中に含まれるフリーのPCR増幅用共通配列プライマー（Forward/Reverse）や酵素などの残留試薬を除去する目的で、PCR PurificationKit(QIAGEN社)を用いて精製する。この溶液をemPCR増幅またはブリッジ増幅適用後、各社(Life Technologies(Solid/Ion Torrent)、Illumina(High Seq), Roche 454)の次世代シーケンサに適用して解析する。 Next, 495 μL of sterilized water, 100 μL of 10 x High Fidelity PCR Buffer (Invitrogen), 2.5 mM dNTP mix 100 μL, 50 mM MgSO 4 40 μL, and 10 μM common primer for PCR amplification (Forward) 100 μL and 10 μM common sequence primer for PCR amplification (Reverse) Mix 100 μL and Platinum Taq Polymerase High Fidelity (Invitrogen) 15 μL, drain the solution filling upper reaction zone 7 and lower reaction zone 8 from outlets 306 and 307, and immediately inject the above solution from inlet 305 did. After that, keep the solution at 94 ° C for 30 seconds, repeat 40 cycles of 3 steps of 94 ° C 30 seconds → 55 ° C 30 seconds → 68 ° C 30 seconds, finally hold 68 ° C for 3 minutes, then cool to 4 ° C A PCR amplification step was performed (FIG. 4 (f)). In order to realize such a temperature cycle, a heat block with heater (aluminum alloy or copper alloy) 309 and a temperature controller 310 may be added. This amplifies the target portion of 20 target genes, but the PCR product size is almost uniform at 200 ± 8 bases. Collect the PCR amplification product solution accumulated in the solution. For the purpose of removing residual reagents such as free PCR amplification common sequence primers (Forward / Reverse) and enzymes contained in this solution, purification is performed using PCR Purification Kit (QIAGEN). After applying this solution to emPCR amplification or bridge amplification, the solution is applied to a next-generation sequencer of each company (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454) and analyzed.
　次に、分子認識タグを用いた増幅バイアスの低減方法について説明する。図6には分子認識領域以外は同じ配列としてシーケンシングされたデータが得られた状態を模式的に示している（得られたシーケンシングデータの関連部分を模式的に図示）。図6中で601、602、603、604、605はランダム配列である分子認識タグ配列も含めて同じ配列であり、それぞれ1、7、4、2、2リードが得られている場合を示している。これらの配列は、図4(e)で2ndストランドが合成された時点ではすべて１分子であり、その後のPCR増幅で分子数が増大すると同時に、異なる分子数になっている。それゆえ、分子認識タグの同じリードは同じ分子としてみなしてよく、すべて１分子とみなされる。その結果、2ndストランドが合成された後の工程のPCR増幅や、溶液を外部に取り出すときに細孔アレイシート内部への吸着による配列ごとに分子数の偏りは、上記同一視によって解消される。 Next, a method for reducing the amplification bias using the molecular recognition tag will be described. FIG. 6 schematically shows a state where data sequenced in the same sequence except for the molecular recognition region is obtained (relevant portions of the obtained sequencing data are schematically shown). In FIG. 6, 601, 602, 603, 604, and 605 are the same sequence including the molecular recognition tag sequence that is a random sequence, and show the cases where 1, 7, 4, 2, and 2 reads are obtained, respectively. Yes. These sequences are all one molecule at the time when the 2nd strand is synthesized in FIG. 4 (e), and the number of molecules increases at the same time as the number of molecules increases by subsequent PCR amplification. Therefore, the same lead of the molecular recognition tag may be regarded as the same molecule, and all are regarded as one molecule. As a result, the unevenness of the number of molecules for each sequence due to PCR amplification in the process after the synthesis of the second strand and adsorption to the inside of the pore array sheet when the solution is taken out is eliminated by the above-mentioned identification.
　ここで、得られたデータとしては、1、7、4、2、2が見かけ上の（分子認識タグ以外は同一の配列の）カウント数になっている。細胞中の分子数はそれぞれ、1カウントと数えなおして合計で5カウント（1、7、4、2、2それぞれが1カウントに対応する）となる。すなわち、分子タグ以外の配列に相当する分子は、増幅前には5分子あったと推定される。実際にデータにおいては、分子認識タグ以外の配列が異なるリードもシーケンシング結果として得られる。このとき、分子認識タグが異なって、他の配列が同じリードをそれぞれ数えることによって、知りたい配列についてのカウントを実行することができる。もともとのサンプルの中には、このカウントに比例する分子数のmRNAが含まれていると推定できる。 Here, as the obtained data, 1, 7, 4, 2, and 2 are apparent counts (the same sequence except for the molecular recognition tag). The number of molecules in each cell is recounted as 1 count, for a total of 5 counts (1, 7, 4, 2, and 2 each corresponding to 1 count). That is, it is estimated that there were 5 molecules corresponding to sequences other than the molecular tag before amplification. Actually, in the data, reads having different sequences other than the molecular recognition tag are also obtained as a sequencing result. At this time, by counting the number of reads in which the molecular recognition tags are different and the other sequences are the same, it is possible to execute the count for the sequence to be known. It can be presumed that the original sample contains mRNA with the number of molecules proportional to this count.
　ここで作製したシートは繰り返し利用可能であり、発現量を知る必要がある遺伝子群については、PCR増幅用共通配列プライマー(Reverse)が付加された遺伝子特異的配列プライマーMix溶液を作製し、上記と同様に2nd cDNA鎖の合成、PCR増幅、およびemPCRを施し、次世代シーケンサにて解析を行えばよい。すなわち、cDNAライブラリを繰り返し利用することによって、高精度な発現分布測定を必要な種類の遺伝子について行うことが可能である。 The prepared sheet can be used repeatedly, and for gene groups that need to know the expression level, a gene-specific sequence primer Mix solution with a common sequence primer (Reverse) for PCR amplification added is prepared. Similarly, synthesis of the 2nd cDNA strand, PCR amplification, and emPCR may be performed and analyzed by a next-generation sequencer. That is, by repeatedly using a cDNA library, it is possible to perform highly accurate expression distribution measurement for a necessary type of gene.
（実施例2）
　本実施例は、アレイ状に配置された細胞群から、個々の細胞の中に含まれるmRNAを、どの細胞由来かについての情報を保持した状態でcDNAライブラリを構築するために、ビーズを用いた核酸トラッピング部を有する核酸抽出デバイスではなく、核酸トラッピング部としてDNAプローブを固定した細孔アレイシートを用いている。また、cDNAライブラリを構築後の核酸増幅としては、PCR増幅ではなくT7プロモータを用いている。 (Example 2)
In this example, beads were used to construct a cDNA library from a group of cells arranged in an array while retaining information on which cells derived mRNA contained in individual cells. Instead of a nucleic acid extraction device having a nucleic acid trapping portion, a pore array sheet having a DNA probe immobilized thereon is used as the nucleic acid trapping portion. Moreover, T7 promoter is used instead of PCR amplification for nucleic acid amplification after constructing the cDNA library.
　本実施例での核酸抽出デバイスの構造とそれを用いた抽出・処理方法を図7、8、9に示した。 The structure of the nucleic acid extraction device and the extraction / treatment method using it in this example are shown in FIGS.
　図7(a)に核酸抽出デバイスの単位構造の断面図を示している。細胞1は上部反応領域7から下部反応領域8に細胞を含むバッファ溶液をデバイスを貫通するように流すことによって細胞トラッピング部2に捕捉される。細胞トラッピング部2はPDMS製のセルアレイデバイス6中である。この細胞捕捉部の直径は細胞の直径よりやや大きい16μmとしている。この直径の設定によって、この細胞トラッピング部に2つ以上の細胞が捕捉されることを防いでいる。核酸トラッピング部である細孔アレイシート71はシートを貫通するような細孔72が多数形成されており、細孔72の内壁にDNAプローブが固定されている。細胞トラップ用のPDMS製のセルアレイデバイス6と核酸トラップ用の細孔アレイシート(多孔質メンブレン)71が核酸抽出デバイスの基本要素である。これら2つの要素が直接重ねられており、両者をつなぐ流路はこれらの要素が役割をかねている。実施例1と同様に、細胞トラップ後、細胞を含むバッファをLysisバッファに置換し、デバイスと垂直方向に電界を印加しながら、細胞を破砕する。これによって、破砕された細胞中のmRNAは細胞トラップ位置の直下の細孔72内壁上のDNAプローブ73にハイブリダイゼーションによって捕捉される。 Fig. 7 (a) shows a sectional view of the unit structure of the nucleic acid extraction device. The cells 1 are captured by the cell trapping unit 2 by flowing a buffer solution containing cells from the upper reaction region 7 to the lower reaction region 8 so as to penetrate the device. The cell trapping unit 2 is in a cell array device 6 made of PDMS. The diameter of the cell trapping part is 16 μm, which is slightly larger than the cell diameter. By setting the diameter, two or more cells are prevented from being trapped in the cell trapping portion. The pore array sheet 71 that is a nucleic acid trapping portion has a large number of pores 72 penetrating the sheet, and a DNA probe is fixed to the inner wall of the pore 72. A cell array device 6 made of PDMS for cell trap and a pore array sheet (porous membrane) 71 for nucleic acid trap are basic elements of the nucleic acid extraction device. These two elements are directly overlapped, and these elements play a role in the flow path connecting them. As in Example 1, after cell trapping, the cell-containing buffer is replaced with Lysis buffer, and the cells are disrupted while an electric field is applied in a direction perpendicular to the device. Thus, mRNA in the disrupted cell is captured by hybridization to the DNA probe 73 on the inner wall of the pore 72 immediately below the cell trap position.
　細胞アレイシート内部に固定されたDNAプローブ73は、5’末端方向からT7プロモータ配列、emPCR増幅用共通配列 (Forward方向)、細胞認識用タグ配列、分子認識用タグ配列、およびオリゴ(dT)配列で構成される。T7プロモータ配列をDNAプローブへ導入する事で、後続のIVT (In Vitro Transcription)によるcRNA83増幅工程（図8(e)）によるターゲット配列の増幅が可能となる。 The DNA probe 73 fixed inside the cell array sheet consists of a T7 promoter sequence from the 5 ′ end direction, a common sequence for emPCR amplification (Forward direction), a cell recognition tag sequence, a molecular recognition tag sequence, and an oligo (dT) sequence. Consists of. By introducing the T7 promoter sequence into the DNA probe, it is possible to amplify the target sequence by the subsequent cRNA83 amplification step (FIG. 8 (e)) by IVT (In Vitro Transcription).
　後述するように、核酸増幅工程において、転写因子によるcDNAからcRNAへの転写反応を行う場合には、DNAプローブは、さらに転写因子のプロモータ配列を含むことが好ましい。そのようなプロモータ配列としては、T7を用いるが他にSP6、T3などが挙げられる。核酸の増幅にはT7RNAポリメラーゼの活性を用いる。 As will be described later, when a transcription reaction from cDNA to cRNA by a transcription factor is performed in the nucleic acid amplification step, the DNA probe preferably further contains a promoter sequence of the transcription factor. As such a promoter sequence, T7 is used, but SP6, T3 and the like are also included. The activity of T7 RNA polymerase is used for nucleic acid amplification.
　本実施例では、T7プロモータ配列を用い、この配列はT7RNAポリメラーゼにより認識され、その下流配列から転写(cRNA83増幅)反応が開始される。 In this example, a T7 promoter sequence is used, this sequence is recognized by T7 RNA polymerase, and a transcription (cRNA83 amplification) reaction is started from the downstream sequence.
　転写因子のプロモータ配列を用いた核酸増幅は等温増幅なため、温度サイクルを加えるための温度制御器が不要になるだけでなく、高温時にデバイス表面に固定したプローブDNAが脱離する可能性を低減することができる。 Nucleic acid amplification using the promoter sequence of transcription factor is isothermal amplification, which not only eliminates the need for a temperature controller to add a temperature cycle, but also reduces the possibility of detachment of probe DNA immobilized on the device surface at high temperatures. can do.
　同様にPCR増幅用共通配列を導入する事で、後続のemPCR増幅工程において共通プライマーとして利用する事ができる。また、細胞認識タグを（例えば5塩基）DNAプローブへ導入する事によって、45=1024個の単一細胞を認識する事が可能となることは実施例1と同様である。さらに、分子認識用タグ配列（例えば7塩基）をDNAプローブへ導入する事により、47=1.6x104分子を認識する事ができるため、次世代シーケンサで得られる膨大な解読データが、どの分子由来であるかを認識する事が可能となることも実施例1と同様である。すわなち、IVT / emPCRなどの増幅工程で生じた遺伝子間の増幅バイアスを修正する事ができるため、始めに試料中に存在していたmRNA量を高い精度で定量する事が可能となる。最も3’側に位置するオリゴ(dT) 配列は、mRNAの3’側に付加されているポリAテールとハイブリダイズし、mRNAを捕捉するために利用される(図7(a))。 Similarly, by introducing a common sequence for PCR amplification, it can be used as a common primer in the subsequent emPCR amplification step. Further, as in Example 1, it is possible to recognize 4 5 = 1024 single cells by introducing a cell recognition tag (for example, 5 bases) into a DNA probe. Furthermore, 4 7 = 1.6x10 4 molecules can be recognized by introducing a molecular recognition tag sequence (for example, 7 bases) into a DNA probe. Similar to the first embodiment, it is possible to recognize the origin. In other words, since the amplification bias between genes generated in the amplification process such as IVT / emPCR can be corrected, the amount of mRNA present in the sample can be quantified with high accuracy. The oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of the mRNA and is used to capture the mRNA (FIG. 7 (a)).
　次に、核酸トラッピング部を構成する細孔アレイシートの作製方法について記す。 Next, a method for producing a pore array sheet constituting the nucleic acid trapping portion will be described.
　細孔アレイシートとしては、陽極酸化法によって作製した市販品を入手可能であり、ここでは孔径200nmで厚さが60μm、13mm角（直径25mmのシートから切断して利用）の細孔アレイシート71を用いた例について説明する。細孔アレイシート71に形成された細孔72は細孔アレイシート71の厚さ方向に貫通しており、細孔同士は完全に独立である。細孔72は流路5の機能もかねる。表面は親水性で、表面への蛋白質の吸着が極めて少なく、酵素反応が効率よく進む。まず、細孔アレイシート71の表面をシランカップリングなどの処理をしてDNAプローブ73を細孔表面に固定する。DNAプローブ73は平均30～100nm2に一個の割合で表面に固定されるので、4～10x106個のDNAプローブが1つの孔に固定される。次いで表面吸着を防止するために表面コート剤で表面をコートする。この表面コートはプローブ固定と同時に行ってもよい。このDNAプローブ密度はこの空間を通過するmRNAをほぼ100％の効率でDNAプローブに捕獲できる密度である。 As the pore array sheet, a commercially available product prepared by an anodic oxidation method is available. Here, a pore array sheet 71 having a pore diameter of 200 nm, a thickness of 60 μm, and a 13 mm square (cut from a 25 mm diameter sheet) is used. An example using this will be described. The pores 72 formed in the pore array sheet 71 penetrate in the thickness direction of the pore array sheet 71, and the pores are completely independent. The pore 72 also functions as the flow path 5. The surface is hydrophilic, the protein adsorption to the surface is extremely small, and the enzymatic reaction proceeds efficiently. First, the surface of the pore array sheet 71 is treated such as silane coupling to fix the DNA probe 73 to the pore surface. Since the DNA probe 73 is fixed to the surface at an average ratio of 30 to 100 nm 2 , 4 to 10 × 10 6 DNA probes are fixed to one hole. Next, the surface is coated with a surface coating agent in order to prevent surface adsorption. This surface coating may be performed simultaneously with the probe fixation. The DNA probe density is such that mRNA passing through this space can be captured by the DNA probe with an efficiency of almost 100%.
　次に、細孔内部へのDNAプローブ固定化方法について詳細に説明する。細孔アレイシート内部の細孔の表面は、高密度にDNAプローブが固定化されると同時に、mRNAやPCR増幅用プライマーなどの核酸、そして逆転写酵素やポリメラーゼなどの蛋白質を吸着しない表面である必要がある。本実施例では、DNAプローブを固定化するためのシランカップリング剤と吸着を防止するシラン化されたMPCポリマーとを適切な割合で同時に細孔表面に共有結合にて固定して、DNAの高密度固定と核酸や蛋白質の安定した吸着抑制を実現した。実際には、まずアルミナ製の細孔アレイシートをエタノール溶液に3分浸漬後、UVO3処理を5分行い、超純水で3回洗浄する。次に平均分子量9700（重合度40）のシラン化MPCポリマーであるMPC0.8-MPTMSi0.2（MPC: 2-Methacryloyloxyethyl phosphorylcholine/MPTMSi: 3-Methacryloxypropyl trimethoxysilane）（例えば、Biomaterials 2009, 30:4930-4938、およびLab Chip 2007, 7:199-206）3mg/mlと0.3mg/mlのシランカップリング剤GTMSi（GTMSi：3-Glycidoxypropyltrimethoxysilane信越化学）、および酸触媒である0.02％酢酸を含む80％エタノール溶液に2時間浸漬した。エタノールで洗浄後、窒素雰囲気で乾燥し、オーブンにて120℃で30分間加熱処理した。次にDNAを固定化するために、1μM 5’アミノ基修飾されたDNAプローブと7.5％グリセロールと0.15M NaClを含む0.05Mホウ酸バッファ（pH 8.5）を細孔アレイシート上にインクジェットプリンタと同じ技術によって、100pLずつ25μm×25μmの領域毎に異なる細胞認識用タグ配列（1024種類）を含むDNAプローブを吐出した。その後、加湿チャンバ内において25℃で2時間反応させた。最後に未反応グリシド基をブロックし、過剰なDNAプローブを除去するために、十分量の10mM Lysと0.01％SDSと0.15M NaClを含むホウ酸バッファ（pH 8.5）で5分間洗浄し、この洗浄液を除去した後、0.01％SDSと0.3M NaClを含む30mMクエン酸ナトリウムバッファ（2xSSC, pH 7.0）を用いて60℃にて洗浄し、過剰DNAを除去した。これによって、DNAプローブの固定と表面処理を完了した。 Next, a method for immobilizing a DNA probe inside the pore will be described in detail. The surface of the pores inside the pore array sheet is a surface that does not adsorb nucleic acids such as mRNA and PCR amplification primers, and proteins such as reverse transcriptase and polymerase at the same time that DNA probes are immobilized at high density. There is a need. In this example, a silane coupling agent for immobilizing a DNA probe and a silanized MPC polymer for preventing adsorption were simultaneously covalently immobilized on the pore surface at an appropriate ratio to increase the DNA We realized density fixation and stable adsorption inhibition of nucleic acids and proteins. Actually, the alumina pore array sheet is first immersed in an ethanol solution for 3 minutes, then treated with UVO3 for 5 minutes, and washed with ultrapure water three times. Next, MPC 0.8 -MPTMSi 0.2 (MPC: 2-Methacryloyloxyethyl phosphorylcholine / MPTMSi: 3-Methacryloxypropyl trimethoxysilane) (eg, Biomaterials 2009, 30: 4930-4938, and Lab Chip 2007, 7: 199-206) 2 in 80% ethanol solution containing 3 mg / ml and 0.3 mg / ml silane coupling agent GTMSi (GTMSi: 3-Glycidoxypropyltrimethoxysilane Shin-Etsu Chemical) and 0.02% acetic acid as acid catalyst. Soaked for hours. After washing with ethanol, it was dried in a nitrogen atmosphere and heat-treated at 120 ° C. for 30 minutes in an oven. Next, in order to immobilize the DNA, 1 μM 5 ′ amino group-modified DNA probe and 0.05 M borate buffer (pH 8.5) containing 7.5% glycerol and 0.15 M NaCl are the same as the inkjet printer on the pore array sheet. Depending on the technique, DNA probes containing different cell recognition tag sequences (1024 types) for each 25 μm × 25 μm region of 100 pL were discharged. Then, it was made to react at 25 degreeC in a humidification chamber for 2 hours. Finally, to wash unreacted glycidic groups and remove excess DNA probe, wash with borate buffer (pH 8.5) containing sufficient 10 mM Lys, 0.01% SDS and 0.15 M NaCl for 5 minutes. Then, the DNA was washed with 60 mM sodium citrate buffer (2 × SSC, pH 7.0) containing 0.01% SDS and 0.3 M NaCl to remove excess DNA. This completed the DNA probe immobilization and surface treatment.
　次に、反応の各ステップを順に説明する。図7(a)に示すようにmRNA74は前実施例と同様にmRNA3’末端のポリA配列に相補的な配列である18塩基のポリT配列によって捕捉する。次に1st cDNA鎖79を合成し、cDNAライブラリを構築する（図7(b)）。次に定量したい遺伝子に対応する複数（~100種）のターゲット遺伝子特異的配列プライマー80を1st cDNA鎖79へアニールさせ（図8(c)）、相補鎖伸長反応により2nd cDNA鎖81を合成させる(図8(d))。すなわちマルチプレックス条件で2nd cDNA鎖合成を行う。これにより、複数のターゲット遺伝子について、増幅用共通配列(Forward/Reverse)を両端に持ち、細胞認識タグ、分子認識タグ、および遺伝子特異的配列がその中に含まれる2本鎖cDNAが合成される。また本実施例では、一例として、20種類(ATP5B、GAPDH、GUSB、HMBS、HPRT1、RPL4、RPLP1、RPS18、RPL13A、RPS20、ALDOA、B2M、EEF1G、SDHA、TBP、VIM、RPLP0、RPLP2、RPLP27、およびOAZ1)の遺伝子特異的配列にターゲット遺伝子のポリAテールから109±8塩基上流部分の20±5塩基を用いたが、これは、後続のIVTによる増幅工程において、増幅産物サイズを約200塩基に統一するためである。PCR産物サイズを統一する事で、煩雑なサイズフラクション精製の工程(電気泳動→ゲルの切り出し→PCR産物の抽出・精製)を除去する事ができ、1分子からの並列増幅（エマルジョンPCRなど）へ直接利用できる効果を持つ。続いて、T7RNAポリメラーゼを細孔中に導入し、cRNA83を合成する（図8(e)）。この過程によって、約1000コピー程度のcRNAが合成される。さらに、emPCRのための2本鎖DNAを合成するために、増幅されたcRNAを鋳型として、PCR増幅用共通配列(Reverse)が付加された複数（~100種）のターゲット遺伝子特異的配列プライマー91をハイブリさせ（図9(f)）、cDNAを合成する（図9(g)）。さらに、前実施例と同様に酵素を用いてcRNAを分解してから、Forward共通プライマーを用いて2ndストランド92を合成することによってemPCR用2本鎖DNA93が合成される（図9(h)）。この増幅産物は、長さがそろっており、そのまま、emPCR、次世代シーケンサにかけることができる。この工程において遺伝子間、ないし分子間で増幅バイアスが生じたとしても、次世代シーケンサデータ取得後に、分子認識タグを利用して増幅バイアスの補正を行う事ができるため、高精度な定量データを得る事が出来ることは前実施例と同様である。 Next, each step of the reaction will be explained in order. As shown in FIG. 7 (a), mRNA 74 is captured by an 18-base poly-T sequence that is complementary to the poly-A sequence at the 3 ′ end of mRNA, as in the previous example. Next, the 1st cDNA strand 79 is synthesized to construct a cDNA library (FIG. 7 (b)). Next, anneal the multiple (~ 100 species) target gene-specific sequence primers 80 corresponding to the gene to be quantified to the 1st cDNA strand 79 (Fig. 8 (c)), and synthesize the 2nd cDNA strand 81 by complementary strand elongation reaction. (FIG. 8 (d)). That is, 2nd cDNA strand synthesis is performed under multiplex conditions. As a result, for a plurality of target genes, a double-stranded cDNA having a common amplification sequence (Forward / Reverse) at both ends and containing a cell recognition tag, a molecular recognition tag, and a gene-specific sequence is synthesized. . In this example, as an example, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ± 5 bases 109 ± 8 bases upstream from the poly A tail of the target gene were used for the gene-specific sequence of OAZ1), and this resulted in an amplification product size of approximately 200 bases in the subsequent IVT amplification step. It is for unifying. By unifying the PCR product size, complicated size fraction purification steps (electrophoresis-> gel excision-> PCR product extraction / purification) can be eliminated, and parallel amplification from one molecule (emulsion PCR, etc.) Has an effect that can be used directly. Subsequently, T7 RNA polymerase is introduced into the pore to synthesize cRNA83 (FIG. 8 (e)). Through this process, about 1000 copies of cRNA are synthesized. Furthermore, in order to synthesize double-stranded DNA for emPCR, using the amplified cRNA as a template, multiple (~ 100 species) target gene-specific sequence primers with a common PCR amplification sequence (Reverse) added. Are hybridized (FIG. 9 (f)) to synthesize cDNA (FIG. 9 (g)). Furthermore, after cRNA is degraded using an enzyme in the same manner as in the previous example, the second strand 92 for emPCR is synthesized by synthesizing the 2nd strand 92 using the forward common primer (FIG. 9 (h)). . This amplification product has the same length and can be directly applied to emPCR and next-generation sequencers. Even if an amplification bias occurs between genes or molecules in this step, high-accuracy quantitative data can be obtained because the amplification bias can be corrected using the molecular recognition tag after acquiring next-generation sequencer data. The thing that can be done is the same as in the previous embodiment.
　次に、図7に記載の核酸抽出デバイスの単位構造を有する図10に示した装置構成で、一連の工程の具体的に記す。図10(b)は図10(a)のA-A’断面であり、図10(c)は図10(a)のB-B’断面に対応する断面図である。細胞インレット308から細胞1を導入して、細胞トラッピング部2に細胞を捕捉し、1st cDNA鎖を合成するまでは実施例1と同様である。1st cDNA鎖を合成した後、85℃にて1.5分保ち逆転写酵素を失活させ、４℃に冷却後、RNaseおよび0.1%Tween20を含む10mM Tris Buffer (pH = 8.0) 10mLをインレット305から注入しアウトレット306、307から排出することによって、RNAを分解し、同量のアルカリ変性剤を同様に流して細孔内の残存物および分解物を除去・洗浄した。続いて、滅菌水690μLと 10 x Ex Taq Buffer (TaKaRa Bio社) 100μLと2.5mM dNTP Mix 100μLと各10μMのPCR増幅用共通配列(Reverse)が付加されたい20種の遺伝子特異的配列プライマー Mix100μLとEx Taq Hot start version (TaKaRa Bio社)10μLを混和し、上部反応領域7および下部反応領域8を満たしている溶液をアウトレット306および307から排出し、直ちに逆転写酵素を含む上記溶液をインレット305から注入した。その後、95℃3分間→44℃2分間→72℃6分間の反応を行い、1st cDNA鎖を鋳型としてプライマーの遺伝子特異的配列をアニールさせた後、相補鎖伸長反応を行い、2nd cDNA鎖を合成させた。 Next, the series of steps will be described specifically with the apparatus configuration shown in FIG. 10 having the unit structure of the nucleic acid extraction device shown in FIG. 10B is a cross-sectional view taken along the line A-A ′ in FIG. 10A, and FIG. 10C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ in FIG. The process is the same as in Example 1 until the cell 1 is introduced from the cell inlet 308, the cell is trapped in the cell trapping part 2, and the 1st cDNA strand is synthesized. After synthesizing the first strand cDNA strand, inactivate the reverse transcriptase for 1.5 minutes at 85 ° C, cool to 4 ° C, and inject 10 mL of 10 mM Tris buffer (pH = 8.0) 含 む 10 mL containing RNase and 0.1% Tween20 from inlet 305 Then, the RNA was decomposed by discharging from the outlets 306 and 307, and the same amount of alkali denaturing agent was flowed in the same manner to remove and wash the residue and decomposition products in the pores. Subsequently, 690 μL of sterilized water, 10 Ex Taq Buffer (TaKaRa Bio) 100 μL, 2.5 mM dNTP Mix 100 μL, and 20 types of gene-specific sequence primers 100 Mix 100 μL to which each 10 μM PCR amplification consensus sequence (Reverse) is added Mix 10 μL of Ex Taq Hot start version (TaKaRa Bio), drain the solution filling upper reaction region 7 and lower reaction region 8 from outlets 306 and 307, and immediately remove the above solution containing reverse transcriptase from inlet 305. Injected. Then, 95 ° C for 3 minutes → 44 ° C for 2 minutes → 72 ° C for 6 minutes, anneal the primer-specific sequence using the 1st cDNA strand as a template, and then perform complementary strand extension reaction to obtain the 2nd cDNA strand. Synthesized.
　続いて、0.1%Tween20を含む10mM Tris Buffer (pH = 8.0) 10mLをインレット305から注入しアウトレット306、307から排出することによって、細孔内の残存物および分解物を除去・洗浄した。さらに、滅菌水340μLとAmpliScribe 10 X Reaction Buffer（EPICENTRE社）100μLと100mM dATP 90μLと100mM dCTP 90μLと100mM dGTP 90μLと100mM dUTP 90μLと100mM DTT、およびAmpliScribe T7 Enzyme Solution（EPICENTRE社）100μLを混和し、上部反応領域7および下部反応領域8を満たしている溶液をアウトレット306および307から排出し、直ちに逆転写酵素を含む上記溶液をインレット305から注入した。その後、溶液の温度を37℃に上げて、180分間保つことによって逆転写反応を完了させ、cRNA増幅を行った。これにより、20種のターゲット遺伝子の目的部分が増幅されるが、いずれもcRNA増幅産物サイズは200±8塩基とほぼ均一である。メンブレンの細孔内部および外部の溶液中に蓄積されたcRNA増幅産物溶液を回収する。この溶液中に含まれる酵素などの残留試薬を除去する目的で、PCR PurificationKit(QIAGEN社)を用いて精製し、50μLの滅菌水に懸濁する。この溶液に、10mM dNTP mix 10μLと50ng/μLのランダムプライマー30μLを混和させ、94℃10秒加熱後0.2℃/秒で温度を30℃まで低下させ、30℃で5分間加熱し、さらに4℃まで低下させる。その後、5xRT Buffer (Invitrogen社)20μLと、0.1M DTT 5μLと、RNase OUT 5μLと、SuperScript III 5μLを混和させ、30℃で５分間加熱し、0.2℃/秒で温度を40℃まで上昇させる。この溶液中に含まれる酵素などの残留試薬を除去する目的で、PCR PurificationKit(QIAGEN社)を用いて精製し、emPCR増幅に適用後、各社(Life Technologies、Illumina, Roche)の 次世代シーケンサに適用して解析する。 Subsequently, 10 mL of 10 mM Tris Buffer (pH = 8.0) containing 0.1% Tween 20 was injected from the inlet 305 and discharged from the outlets 306 and 307, thereby removing and washing the residue and decomposition products in the pores. Furthermore, sterilized water 340μL and AmpliScribe 10 X Reaction Buffer (EPICENTRE) 100μL and 100mMAdATP 90μL and 100mM dCTP 90μL and 100mM dGTP と 90μL and 100mM dUTP 90μL and 100mM DTT, and AmpliSzybe The solution filling the upper reaction region 7 and the lower reaction region 8 was discharged from the outlets 306 and 307, and the solution containing reverse transcriptase was immediately injected from the inlet 305. Thereafter, the temperature of the solution was raised to 37 ° C. and maintained for 180 minutes to complete the reverse transcription reaction, and cRNA amplification was performed. As a result, target portions of 20 target genes are amplified, and the size of the cRNA amplification product is almost uniform at 200 ± 8 bases. Collect the cRNA amplification product solution accumulated in the solution inside and outside the pores of the membrane. For the purpose of removing residual reagents such as enzymes contained in this solution, it is purified using PCR® Purification Kit (QIAGEN) and suspended in 50 μL of sterile water. To this solution, mix 10 μL of 10 mM dNTP mix and 30 μL of 50 ng / μL random primer, heat at 94 ° C. for 10 seconds, decrease the temperature to 30 ° C. at 0.2 ° C./second, heat at 30 ° C. for 5 minutes, and further increase to 4 ° C. To lower. Then, mix 5 μL RT Buffer (Invitrogen) 20 μL, 0.1 M DTT 5 μL, RNase OUT 5 μL, SuperScript III III 5 μL, heat at 30 ° C. for 5 minutes, and raise the temperature to 40 ° C. at 0.2 ° C./second. For the purpose of removing residual reagents such as enzymes contained in this solution, it is purified using PCRificationPurificationKit (QIAGEN), applied to emPCR amplification, and then applied to next-generation sequencers of each company (Life Technologies, Illumina, Roche) And analyze.
（実施例3）
　１細胞ごとの遺伝子解析を実現する核酸抽出デバイスによって、細胞の個性／状態を識別することが可能である。一方、非侵襲顕微鏡観察では細胞を生かしたままで細胞の形態や化学組成を計測することが可能である。しかし、顕微鏡イメージのみの情報から細胞の状態を識別することは、細胞の個性／状態が多様で不安定であるため、極めて困難であった。本実施例では、１細胞ごとの遺伝子解析による細胞個性の識別と非侵襲イメージングを組み合わせるためのデバイスと装置構成を示す。図3(a)や図7(a)のようなデバイス構造をもったものを用いて細胞を捕捉した状態で顕微鏡観察する場合、ビーズや細孔シートが透明な材料でできていたとしても、その材料の屈折率が溶液の屈折率と一般に異なるため、励起光や照明光が散乱されて、解像度の低下や背景光レベルの上昇という問題が生じる。本実施例では図2の構成を典型例とした核酸抽出デバイスと光学顕微鏡を組み合わせた例を示す。 (Example 3)
It is possible to identify the individuality / state of a cell by a nucleic acid extraction device that realizes gene analysis for each cell. On the other hand, in non-invasive microscope observation, it is possible to measure cell morphology and chemical composition while keeping cells alive. However, it is extremely difficult to distinguish the state of a cell from information only from a microscopic image because the individuality / state of the cell is various and unstable. In this example, a device and an apparatus configuration for combining identification of cell individuality by gene analysis for each cell and noninvasive imaging are shown. When microscopic observation is performed with cells captured using the device structure shown in FIG. 3 (a) or FIG. 7 (a), even if beads and pore sheets are made of a transparent material, Since the refractive index of the material is generally different from the refractive index of the solution, excitation light and illumination light are scattered, causing problems such as a decrease in resolution and an increase in background light level. In the present embodiment, an example in which a nucleic acid extraction device and an optical microscope are combined with the configuration of FIG. 2 as a typical example is shown.
　図11に本実施例での核酸抽出デバイスの構造および装置を示す。図11(b)は図11(a)のA-A’断面であり、図11(c)は図11(a)のB-B’断面に対応する断面図である。本例ではPDMS製の基板6の形状を工夫して、細胞の下方ではあるが直下には核酸トラッピング部を配置せず、周辺のリング状の領域を設けこの部分に磁性ビーズ12をパッキングした。基板平面に垂直方向に電界を印加することによってmRNAなどの核酸はリング状にパッキングされたビーズ部分を電気泳導によって導かれビーズ表面のDNAプローブによって捕捉される。以下のプロセスは実施例1の場合と同様である。PDMSで作製した細胞トラッピング部の直径は16μm、細胞トラッピング部の直下の顕微鏡窓1101の直径は25μm高さは15μmとした。また、細孔アレイシート35についても、細胞トラッピング部直下に対応する部分は細孔が形成されないように、陽極酸化時にレジストマスクによって保護している。なお、陽極酸化のパターニングを行わず、顕微鏡窓1101の厚さを顕微鏡光学系のデバイス面に垂直方向の焦点深度よりも厚くすることによって、細孔アレイシートによる散乱の影響を小さくすることも可能である。 FIG. 11 shows the structure and apparatus of the nucleic acid extraction device in this example. FIG. 11B is a cross-sectional view taken along the line A-A ′ of FIG. 11A, and FIG. 11C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ of FIG. In this example, the shape of the substrate 6 made of PDMS was devised so that a nucleic acid trapping portion was not arranged just below the cells but directly below, and a peripheral ring-shaped region was provided and the magnetic beads 12 were packed in this portion. By applying an electric field in the direction perpendicular to the substrate plane, nucleic acids such as mRNA are guided by the electrophoretic guide to the bead portion packed in a ring shape and captured by the DNA probe on the bead surface. The following process is the same as that in Example 1. The diameter of the cell trapping part prepared by PDMS was 16 μm, the diameter of the microscope window 1101 immediately below the cell trapping part was 25 μm, and the height was 15 μm. The pore array sheet 35 is also protected by a resist mask at the time of anodization so that pores are not formed in the portion corresponding to the portion immediately below the cell trapping portion. It is also possible to reduce the influence of scattering by the pore array sheet by making the thickness of the microscope window 1101 thicker than the depth of focus perpendicular to the device surface of the microscope optical system without performing anodization patterning. It is.
　また、図12に示すように、核酸トラッピング部を細胞の周囲ではなく、横に配置することも可能である。図12(b)は図12(a)のA-A’断面であり、図12(c)は図12(a)のB-B’断面に対応する断面図である。この例では1201が核酸トラッピング部である。この場合もビーズ上にDNAプローブを固定し、mRNAを捕捉する。他のデバイスの構成およびサンプル調整方法は実施例1と同様である。 Also, as shown in FIG. 12, it is possible to arrange the nucleic acid trapping portion not on the periphery of the cell but on the side. 12B is a cross-sectional view taken along the line A-A ′ in FIG. 12A, and FIG. 12C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ in FIG. In this example, 1201 is a nucleic acid trapping unit. In this case as well, a DNA probe is immobilized on the beads and mRNA is captured. Other device configurations and sample adjustment methods are the same as in the first embodiment.
　また、図11、12ではビーズをパッキングした領域を核酸トラッピング部としたが、細孔アレイシートを核酸トラッピング部とする実施例2と同様に、細孔を形成する部分を制限することによって、図11および12に示したものと同様の構成をビーズを用いずに作製することが可能であることもいうまでもない。 In addition, in FIGS. 11 and 12, the region packed with beads is used as a nucleic acid trapping portion, but in the same manner as in Example 2 in which the pore array sheet is used as a nucleic acid trapping portion, the portion where pores are formed is limited. Needless to say, it is possible to produce a configuration similar to that shown in 11 and 12 without using beads.
　このような核酸抽出デバイスを用いて、遺伝子発現解析のために細胞を破砕して詳細な遺伝子発現解析を行うまえに、高解像度な細胞が生きた状態での形状や蛍光染色による遺伝子や蛋白質の定量またはラマンイメージングを行い、これらのイメージングデータと遺伝子発現解析データを対応させることが可能となる。これを実現するためのシステム構成について以下で説明する。 Using such a nucleic acid extraction device, before performing detailed gene expression analysis by crushing cells for gene expression analysis, the shape and fluorescence staining of genes and proteins in high-resolution cells are alive. Quantitative or Raman imaging can be performed, and these imaging data can be associated with gene expression analysis data. A system configuration for realizing this will be described below.
　まず、図13に核酸抽出デバイスを構築するために平面状のデバイス（セルアレイ1320と細孔アレイシート1321など）の上に配置した細胞サンプルについて、光学顕微鏡による計測と上記デバイスを用いた遺伝子発現解析結果を個々の細胞のデータについて対応させることによって細胞の動態を詳細に計測するための最小のシステム構成を示す。 First, in order to construct a nucleic acid extraction device in FIG. 13, cell samples placed on a planar device (cell array 1320, pore array sheet 1321, etc.) are measured with an optical microscope and gene expression analysis using the above device. The minimum system configuration for measuring cell dynamics in detail by matching the results with individual cell data is shown.
　1200は核酸抽出デバイスおよびそのデバイス上に配置された細胞サンプルを表している。1201は図11に代表される細胞からのmRNA抽出と核酸増幅を行うためのフローシステムである。このフローシステムの中で、細胞由来のmRNAを処理することによって、次世代（大規模）DNAシーケンサ1205で配列を決定するのに必要な量で一定の長さを持ち、末端に核酸処理前の情報を記したタグ配列を含む増幅産物を得る。ここでも、矢印1211は増幅産物の移動を示している。一方、デバイス上の細胞は事前に細胞のデバイス上での位置を特定した形で光学顕微鏡1203による観察を行う。ここで細い矢印は情報の移動を示す。光学顕微鏡1203としては位相差顕微鏡、微分干渉顕微鏡、蛍光顕微鏡、レーザ―走査共焦点蛍光顕微鏡、ラマン顕微鏡、非線形ラマン顕微鏡（CARS顕微鏡、SRS顕微鏡,RIKE顕微鏡）、IR顕微鏡等を含む。これらの光学顕微鏡で得られる情報は遺伝子情報に関する限り、得られる情報が少ない。しかし、基本的には細胞が生きた状態での計測が可能であり、細胞の経時変化を計測することができ、刺激に対する細胞の応答をリアルタイムに計測することができる。デバイス上での位置情報を保存したデバイスを利用することによって、遺伝子発現に関する詳細情報と顕微鏡による時間変化を含む情報を対応させることができる。このことを実現するためには、次世代（大規模）DNAシーケンサからの配列情報1212と光学顕微鏡像の情報1213とタグ配列と対応させた位置情報1214を統合する情報システム1206をシステム内に設ける必要がある。本発明において、上記で説明した細胞の計測情報を統合するシステムの最小の構成は次世代（大規模）DNAシーケンサ1205以外の部分のシステム1207であり、DNAシーケンサとの情報の入力とサンプル（核酸増幅産物）の出力を持っているものである。 1200 represents a nucleic acid extraction device and a cell sample placed on the device. 1201 is a flow system for performing mRNA extraction and nucleic acid amplification from cells represented by FIG. In this flow system, by processing cell-derived mRNA, it has a certain length in order to determine the sequence with the next-generation (large-scale) DNA sequencer 1205, and the end is processed before nucleic acid processing. An amplification product containing the tag sequence with information is obtained. Again, arrow 1211 indicates the movement of the amplification product. On the other hand, the cells on the device are observed by the optical microscope 1203 in a form in which the positions of the cells on the device are specified in advance. Here, a thin arrow indicates movement of information. The optical microscope 1203 includes a phase contrast microscope, a differential interference microscope, a fluorescence microscope, a laser-scanning confocal fluorescence microscope, a Raman microscope, a nonlinear Raman microscope (CARS microscope, SRS microscope, RIKE microscope), an IR microscope, and the like. As far as genetic information is concerned, there is little information obtained with these optical microscopes. However, basically, the measurement can be performed in a state where the cells are alive, the change with time of the cells can be measured, and the response of the cells to the stimulus can be measured in real time. By using a device that stores position information on the device, detailed information related to gene expression can be associated with information including temporal changes by a microscope. In order to realize this, an information system 1206 is provided in the system that integrates sequence information 1212 from the next generation (large-scale) DNA sequencer, optical microscope image information 1213, and position information 1214 corresponding to the tag sequence. There is a need. In the present invention, the minimum configuration of the system that integrates the cell measurement information described above is a system 1207 of a portion other than the next generation (large-scale) DNA sequencer 1205. Information input to the DNA sequencer and a sample (nucleic acid) (Amplification product) output.
　図13に光学顕微鏡として蛍光顕微鏡を組み合わせた場合のシステム構成例を示す。1203は蛍光顕微鏡である。細胞1中には計測したい蛋白室（例えばp53）にGFPを発現させるか、免疫染色によって、特定の蛋白に蛍光体を導入している。このようにして得られる個々の細胞中の発現蛋白質量のデータは、細胞破砕後核酸抽出デバイス中でサンプル処理されて、DNAシーケンシングによって定量されることによって得られる遺伝子発現データと細胞ごとに相関を取ることができる。このとき細胞を認識するために、DAPIによって核酸を染色し、細胞核を認識することによって、細胞位置を蛍光顕微鏡によって識別している。蛋白質量はGFPの発現で計測しているため、経時変化を追うことができるが、同時に計測可能な蛋白質の種類は数種類程度である。一方、シーケンシングによる遺伝子発現解析は一度に100程度は可能であり、プローブを用意すれば1000種類の解析も可能である。それゆえ、細胞内の詳細な遺伝子発現制御に関する情報が個々の細胞ごとに得られる。しかし、これについては経時変化をとらえることができない。しかし、両者を組み合わせることによって、どのような蛋白質発現のときにどのような遺伝子発現であるかというデータが事前に得られていれば、蛋白質の発現データのみから、遺伝子制御に関する情報を推定することが可能となる。このような、蛍光顕微鏡データと遺伝子発現データの対応付けと遺伝子制御に関する情報の推定を情報システム1206で実行する。 Fig. 13 shows a system configuration example when a fluorescence microscope is combined as an optical microscope. 1203 is a fluorescence microscope. In cell 1, GFP is expressed in a protein chamber (for example, p53) to be measured, or a fluorescent substance is introduced into a specific protein by immunostaining. The amount of protein expressed in individual cells is correlated with the gene expression data obtained by processing the sample in a nucleic acid extraction device after cell disruption and quantifying it by DNA sequencing. Can take. At this time, in order to recognize the cell, the nucleic acid is stained with DAPI and the cell nucleus is recognized, and the cell position is identified by a fluorescence microscope. Since the protein mass is measured by the expression of GFP, the change with time can be followed, but there are several types of proteins that can be measured simultaneously. On the other hand, gene expression analysis by sequencing can be about 100 at a time, and 1000 types of analysis are possible if a probe is prepared. Therefore, information on detailed gene expression control in the cell can be obtained for each individual cell. However, it is impossible to capture the change with time. However, if the data on what kind of protein expression is obtained in advance by combining the two, information about gene regulation should be estimated from only the protein expression data. Is possible. Information system 1206 executes such an association of fluorescence microscope data and gene expression data and estimation of information related to gene control.
　次に蛍光顕微鏡1203の構成の詳細を示す。1300は光源であり、ここでは水銀ランプである。1301は励起波長を決める励起フィルタ、1302はダイクロイックミラー、1303は受光波長を選択するエミッションフィルタである。細胞に複数種類の蛍光体が導入されて、同時に計測するとき、1301、1302、1303を制御1304によって選択し、特定の蛍光体からの光のみを計測するようにする。細胞の蛍光イメージは対物レンズ1305、結像レンズ1306、CCDカメラ1307によって行う。これらを制御、画像データ取得を行う制御コンピュータは1308である。 Next, the details of the configuration of the fluorescence microscope 1203 are shown. 1300 is a light source, here a mercury lamp. 1301 is an excitation filter that determines the excitation wavelength, 1302 is a dichroic mirror, and 1303 is an emission filter that selects a light reception wavelength. When a plurality of types of phosphors are introduced into a cell and simultaneously measured, 1301, 1302, and 1303 are selected by the control 1304, and only light from a specific phosphor is measured. The fluorescence image of the cell is obtained by an objective lens 1305, an imaging lens 1306, and a CCD camera 1307. 1308 is a control computer for controlling these and acquiring image data.
　次にフローシステム1201の制御系について説明する。フローシステムの制御コンピュータは1309であり、これはＸＹステージ1310を制御して、顕微鏡像を移動させる。このとき、制御コンピュータ上には細孔アレイシート上の位置座標と細胞認識タグの配列データおよびＸＹステージ位置座標を用いて計算される、顕微鏡像上の位置座標を対応させることができる。この制御コンピュータ1309は細胞のフローセルシステムへの細胞導入を制御する細胞導入制御装置1311、細胞の状態を変化させる分化誘導剤や細胞の応答を調べたい薬剤や、細胞を破砕するためのライセートやサンプル処理のための試薬の導入を制御する試薬制御装置1312、細胞培養条件、PCR時の温度サイクルを制御する温度、CO2濃度制御装置1313、不要な試薬や、細胞、培地交換などに用いる上部試薬排出装置1314、調製された核酸増幅産物を排出するための下部試薬排出装置1315を適切に制御している。最終的に得られた核酸増幅産物は次世代（大規模）DNAシーケンシングシステム1205に渡され配列解析を行う。このときシーケンシングのためのemPCRやブリッジアンプはこのシステムの中で実行されるものとしている。制御コンピュータ上で照合された画像の位置情報と細胞認識タグ配列情報は統合情報システム1206に送られ、蛍光イメージから得られる蛋白量と遺伝子発現量の対応付けを行う。さらに同じシステムで遺伝子発現解析データの経時変化推定を実行する。これによって、遺伝子発現ネットワークのダイナミクスを計測することが可能となる。 Next, the control system of the flow system 1201 will be described. The control computer of the flow system is 1309, which controls the XY stage 1310 to move the microscope image. At this time, the position coordinates on the microscopic image calculated using the position coordinates on the pore array sheet, the array data of the cell recognition tag and the XY stage position coordinates can be made to correspond on the control computer. This control computer 1309 is a cell introduction control device 1311 that controls the introduction of cells into the flow cell system, a differentiation inducing agent that changes the state of the cell, a drug that wants to investigate the response of the cell, a lysate or sample for crushing the cell Reagent control device 1312 that controls the introduction of reagents for processing, cell culture conditions, temperature that controls the temperature cycle during PCR, CO 2 concentration control device 1313, unnecessary reagents and upper reagents used for cells, medium replacement, etc. The discharge device 1314 and the lower reagent discharge device 1315 for discharging the prepared nucleic acid amplification product are appropriately controlled. The finally obtained nucleic acid amplification product is passed to the next generation (large-scale) DNA sequencing system 1205 for sequence analysis. At this time, emPCR and bridge amplifier for sequencing are assumed to be executed in this system. The positional information of the image and the cell recognition tag sequence information collated on the control computer are sent to the integrated information system 1206 to associate the protein amount obtained from the fluorescence image with the gene expression amount. Furthermore, the temporal change estimation of gene expression analysis data is executed with the same system. This makes it possible to measure the dynamics of the gene expression network.
　また、この蛍光顕微鏡は細胞内の計測のみならず、細孔アレイシート中に導かれ、抗体によって捕捉サイトカイン等の細胞から分泌される物質を免疫蛍光染色してその量を計測するために用いてもよい。もちろん、同様にして、破砕後の遺伝子発現量の解析に用いてもよい。 This fluorescent microscope is used not only for intracellular measurement, but also for immunofluorescent staining of substances secreted from cells, such as captured cytokines, which are guided into the pore array sheet and captured by antibodies, and measure the amount thereof. Also good. Of course, the gene expression level after disruption may be used in the same manner.
　図14に蛍光顕微鏡の代わりに微分干渉顕微鏡を組み合わせた例を示す。微分干渉顕微鏡像は蛍光試薬を用いず形状を計測するのみであるが、再生医療など体内に細胞を戻さなくてはならない場合にもっとも細胞への影響度が小さい計測方法の一つである。この画像から得られる細胞形状の変化と遺伝子発現の変化の間に対応が付けることができる場合はもっとも、細胞ダメージが少なく、詳細な細胞分類ができる計測システムとなる。 Fig. 14 shows an example in which a differential interference microscope is combined instead of a fluorescence microscope. The differential interference microscope image only measures the shape without using a fluorescent reagent, but it is one of the measurement methods that has the least influence on cells when cells must be returned to the body, such as in regenerative medicine. When a correspondence can be made between the change in the cell shape obtained from this image and the change in the gene expression, the measurement system is capable of performing detailed cell classification with little cell damage.
　1401は光源でここではハロゲンランプである。1402は偏光子、1403、1404はそれぞれWollastonフィルタおよびWollastonプリズムである。1405はコンデンサレンズ、1406は対物レンズである。 1401 is a light source, here a halogen lamp. 1402 is a polarizer, 1403 and 1404 are a Wollaston filter and a Wollaston prism, respectively. 1405 is a condenser lens, and 1406 is an objective lens.
　図15に光学顕微鏡としてCARS顕微鏡を用いた例を示す。CARS顕微鏡はラマン顕微鏡やIR顕微鏡と同様にレーザ励起部分の化学種に対応したスペクトルが得られるため、微分干渉顕微鏡よりも細胞状態に対する情報量を増大させることができる。ただし、かつCARSは非線形過程で、ラマン信号にくらべて、信号強度が強く、比較的弱いレーザ励起強度で十分なシグナルを得ることができるため、細胞へのダメージが小さいというメリットがある。このようなCARSイメージと遺伝子発現解析データを対応させることによって、より詳細な細胞状態の判定が可能となる。 FIG. 15 shows an example using a CARS microscope as an optical microscope. The CARS microscope, like the Raman microscope and IR microscope, can obtain a spectrum corresponding to the chemical species of the laser excitation part, and therefore can increase the amount of information on the cell state than the differential interference microscope. However, CARS is a non-linear process, and has a merit that damage to cells is small because signal intensity is strong compared to Raman signal and sufficient signal can be obtained with relatively weak laser excitation intensity. By correlating such CARS image and gene expression analysis data, it becomes possible to determine the cell state in more detail.
　1501は光源でここではパルスレーザ（マイクロチップレーザ）である。これをビームスプリッタ1502にて２つに分波し、一方を非線形ファイバ（フォトニッククリスタルファイバ）1503に導入し、ストークス光を生成する。もう一方の光はそのままポンプ光およびプローブ光として用いて、サンプル（細胞中）に水浸対物レンズ1504で集光してアンチストークス光を生成する。ハイパスフィルタ1505にてアンチストークス光のみを透過させて、分光器1506を通して、分光器用CCDカメラ1507でコヒーレントアンチストークスラマンスペクトルを取得する。 1501 is a light source, here a pulse laser (microchip laser). This is split into two by a beam splitter 1502, and one is introduced into a nonlinear fiber (photonic crystal fiber) 1503 to generate Stokes light. The other light is used as it is as pump light and probe light, and is condensed on the sample (in the cell) by the water immersion objective lens 1504 to generate anti-Stokes light. Only the anti-Stokes light is transmitted through the high-pass filter 1505, and the coherent anti-Stokes Raman spectrum is acquired through the spectroscope 1506 by the CCD camera 1507 for the spectroscope.
（実施例4）
　本実施例は細胞トラッピング部が細胞と同程度の大きさの開口部で構成されているのではなく、細胞表面を化学的に捕捉する物質、すなわち細胞表面の物質に化学的に結合する物質を固定した領域で構成されている例を示す。実施例1（図1）に対応して細胞トラッピング部を変更した例を図16に示す。核酸トラッピング（捕捉）に用いたビーズの上の一部の領域に細胞表面と結合する抗体などの蛋白質を固定したビーズを配置している。ビーズ上の抗体は特定の種類の細胞を捕捉するという機能を付加することができる。例えばCD（cluster of differentiation）抗体と呼ばれる一群の抗体は白血球を中心とする細胞の膜蛋白の種類に対応する抗体群がある。この抗体をビオチン化してビーズ上のストレプトアビジンで固定化して、図16の細胞トラッピング部2上にインクジェットプリンタ技術を用いて打ち込むことによって、特定のCD分類の抗原を持つ細胞を捕捉することが可能となる。もちろん、CD抗体を直接ビーズ上に固定するのではなく、ビオチン修飾した2次抗体を介してビーズ上に固定してもよいことはいうまでもない。また、CD抗体以外の抗体をビーズ上に固定してもよいし、細胞上の受容体に結合する分子を固定してもよい。このような分子の例としてはフィブロネクチンがある。フィブロネクチンは細胞上のインテグリンと結合することが知られている。フィブロネクチンをビーズ上に固定することによって、接着性の細胞を捕捉することが可能となる。 (Example 4)
In this embodiment, the cell trapping portion is not composed of an opening having the same size as the cell, but a substance that chemically captures the cell surface, that is, a substance that chemically binds to the cell surface substance. An example composed of fixed areas is shown. FIG. 16 shows an example in which the cell trapping unit is changed corresponding to Example 1 (FIG. 1). Beads to which proteins such as antibodies that bind to the cell surface are immobilized are arranged in a partial region on the beads used for nucleic acid trapping (capture). The antibody on the bead can add a function of capturing a specific type of cell. For example, a group of antibodies called CD (cluster of differentiation) antibodies includes an antibody group corresponding to the type of cell membrane protein centering on leukocytes. This antibody can be biotinylated, immobilized with streptavidin on the beads, and captured by using the inkjet printer technology on the cell trapping part 2 in Fig. 16 to capture cells with a specific CD classification antigen. It becomes. Of course, it is needless to say that the CD antibody is not directly immobilized on the beads, but may be immobilized on the beads via a biotin-modified secondary antibody. Further, antibodies other than CD antibodies may be immobilized on beads, or molecules that bind to receptors on cells may be immobilized. An example of such a molecule is fibronectin. Fibronectin is known to bind to integrins on cells. By fixing fibronectin on the beads, it becomes possible to capture adherent cells.
　細胞表面の物質に化学的に結合する物質のその他の例としては、細胞外マトリックス、例えば、コラーゲン、ラミニン、エラスチンなどが挙げられる。 Other examples of substances that chemically bind to substances on the cell surface include extracellular matrices such as collagen, laminin, and elastin.
　次に、実施例3（図2）に対応するデバイス構成において細胞捕捉を化学的に行う例を示す。核酸抽出デバイスの透明領域の一部に細胞を捕捉するための物質を固定する例である。捕捉用物質は上記と同様抗体を用いてもよいし、他の物質を用いてもよい。図17に核酸抽出デバイスの構成例を示す。細胞補足用抗体1702を細胞トラッピング部2に固定している。固定方法はビオチン化した抗体とストレプトアビジンをこの領域に固定することによっている。抗体に対応する抗原1703を持つ細胞のみを捕捉することが可能となる。 Next, an example in which cell capture is chemically performed in the device configuration corresponding to Example 3 (FIG. 2) will be described. This is an example in which a substance for capturing cells is fixed to a part of a transparent region of a nucleic acid extraction device. As the capture substance, an antibody may be used as described above, or another substance may be used. FIG. 17 shows a configuration example of the nucleic acid extraction device. Cell capturing antibody 1702 is fixed to cell trapping section 2. The immobilization method is by immobilizing biotinylated antibody and streptavidin in this region. Only cells having the antigen 1703 corresponding to the antibody can be captured.
　さらに、実施例2（図7）に対応する例を示す。図18に示すように細孔アレイシート71上の細胞を捕捉するための領域1801に抗体を固定することによって、抗体に対応する抗原1802を有する細胞を補足することが可能な細胞トラッピング部を形成している。抗体を特定の位置に固定するために、インクジェットプリンタヘッドを用いて、ビオチン化した抗体を数十pL程度ずつ打ち込んだ。実施例3で示した方法によってデバイス表面全体はストレプトアビジンがコートされているので、特定の領域にのみ抗体が固定されることになる。 Furthermore, an example corresponding to Example 2 (FIG. 7) is shown. As shown in Fig. 18, by immobilizing the antibody in the region 1801 for capturing cells on the pore array sheet 71, a cell trapping part capable of capturing cells having the antigen 1802 corresponding to the antibody is formed. is doing. In order to fix the antibody at a specific position, about a few tens of pL of biotinylated antibody was injected using an inkjet printer head. Since the entire device surface is coated with streptavidin by the method shown in Example 3, the antibody is immobilized only in a specific region.
（実施例5）
　実施例1～4では2ndストランド形成の工程（図2(d)-(e)および図8(c)-(d)で示した工程）およびPCR増幅の工程（図2(f)および図8(e)）も図5、図10、図11または図12に示したような装置の中で行う例を示した。しかし、核酸抽出デバイス1901の全部または核酸抽出デバイスを複数のデバイスに分割したものを樹脂製のチューブ1902（一般に用いられる0.2mLや1.5mL容量などのチューブ）または96穴や384穴プレートにデバイスを図19のように挿入し、2ndストランド合成およびPCR増幅に必要な試薬1903をこのチューブの中に混和することによって行ってもよい。このような構成にすることによってこれらの工程の条件についてユーザーが自由に条件を変更することが可能であるばかりでなく、2ndストランド合成からチューブの中で行うことによって、細胞認識タグや分子認識タグを実施例1-4に示した位置とは反対側の末端に挿入することが可能となる。 (Example 5)
In Examples 1 to 4, the second strand formation step (steps shown in FIGS. 2 (d)-(e) and 8 (c)-(d)) and the PCR amplification step (FIG. 2 (f) and FIG. 8). (e)) also shows an example performed in the apparatus as shown in FIG. 5, FIG. 10, FIG. 11 or FIG. However, the entire nucleic acid extraction device 1901 or the nucleic acid extraction device divided into multiple devices can be placed in a resin tube 1902 (commonly used 0.2 mL or 1.5 mL capacity tubes) or 96 or 384-well plates. Insertion as shown in FIG. 19 may be performed by mixing the reagent 1903 necessary for 2nd strand synthesis and PCR amplification into this tube. With this configuration, not only can the user freely change the conditions for these processes, but the cell recognition tag and molecular recognition tag can be changed from the 2nd strand synthesis in the tube. Can be inserted at the end opposite to the position shown in Example 1-4.
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated into the present specification as they are.
　本発明によって、生体分子の定量、配列決定、分子同定が、多数の培養細胞や多数の免疫細胞や（血中）がん細胞等に対して実行でき、どのような状態にある細胞群がどの程度の数だけ生体に存在するかを計測することが可能となる。これは、がんなどの早期の診断やiPS細胞のヘテロジェナイエティを計測することも可能となる。 According to the present invention, quantification, sequencing, and molecular identification of biomolecules can be performed on a large number of cultured cells, a large number of immune cells, (in blood) cancer cells, and the like. It is possible to measure whether or not a certain number exists in the living body. This makes it possible to measure early diagnosis of cancer and the heterogeneity of iPS cells.
1：細胞
2：細胞トラッピング部
3：流路
4：核酸トラッピング部
5：流路
6：平面基板
7：上部反応領域
8：下部反応領域 1: Cell
2: Cell trapping section
3: Flow path
4: Nucleic acid trapping section
5: Flow path
6: Flat substrate
7: Upper reaction area
8: Lower reaction area

Claims (14)

  1. 　それぞれ１つの細胞を固定するための細胞トラッピング部と、
    　前記細胞から核酸を抽出するための核酸抽出液が前記細胞トラッピング部を通過して上から下へ流れる流路と、
    　前記流路を介して前記細胞トラッピング部と繋がり前記細胞トラッピング部より下方に配置され、抽出された核酸を固定する核酸トラッピング部と、
    　核酸抽出後の溶液を前記核酸トラッピング部から前記細胞トラッピング部とは反対側に排出する流路とを備え、
    　前記細胞トラッピング部と、前記２つの流路と、前記核酸トラッピング部が上下方向に対を形成し、この対が、平面方向に複数配置されている、ことを特徴とする核酸抽出デバイス。     A cell trapping part for fixing one cell each;
    A flow path through which the nucleic acid extract for extracting nucleic acid from the cells flows from the top to the bottom through the cell trapping section;
    A nucleic acid trapping section that is connected to the cell trapping section via the flow path and is arranged below the cell trapping section and fixes the extracted nucleic acid;
    A flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping part to the opposite side of the cell trapping part,
    A nucleic acid extraction device, wherein the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction.
  2. 　前記核酸トラッピング部が、核酸トラップのためのDNAが固定されたビーズを含むことを特徴とする請求項１記載の核酸抽出デバイス。 The nucleic acid extraction device according to claim 1, wherein the nucleic acid trapping section includes beads on which DNA for nucleic acid trap is fixed.
  3. 　前記核酸トラッピング部が、細孔に核酸トラップのためのDNAが固定された多孔質メンブレンを含むことを特徴とする請求項１記載の核酸抽出デバイス。 The nucleic acid extraction device according to claim 1, wherein the nucleic acid trapping portion includes a porous membrane in which DNA for nucleic acid trap is fixed in a pore.
  4. 　前記細胞トラッピング部に、細胞表面の物質に化学的に結合する物質が固定されていることを特徴とする請求項１～３のいずれか１項記載の核酸抽出デバイス。 The nucleic acid extraction device according to any one of claims 1 to 3, wherein a substance that chemically binds to a cell surface substance is fixed to the cell trapping portion.
  5. 　前記核酸トラップのためのDNAの一部がチップ上の位置を特定するための配列を含むことを特徴とする請求項２または３記載の核酸抽出デバイス。 The nucleic acid extraction device according to claim 2 or 3, wherein a part of DNA for the nucleic acid trap includes a sequence for specifying a position on the chip.
  6. 　前記核酸トラップのためのDNAの一部がトラップした核酸分子ごとに異なる配列を含むことを特徴とする請求項２または３記載の核酸抽出デバイス。 The nucleic acid extraction device according to claim 2 or 3, wherein a part of DNA for the nucleic acid trap contains a different sequence for each trapped nucleic acid molecule.
  7. 　前記核酸トラッピング部にトラップしたRNAを逆転写するための酵素を導入する手段を有することを特徴とする請求項６記載の核酸抽出デバイス。 The nucleic acid extraction device according to claim 6, further comprising means for introducing an enzyme for reverse transcription of RNA trapped in the nucleic acid trapping section.
  8. 　前記細胞トラッピング部の直下が光学的に透明な材料で構成されていることを特徴とする請求項１～７のいずれか１項記載の核酸抽出デバイス。 The nucleic acid extraction device according to any one of claims 1 to 7, wherein a portion immediately below the cell trapping portion is made of an optically transparent material.
  9. 　前記細胞トラッピング部の直下に核酸トラッピング部を設けたことを特徴とする請求項１～８のいずれか１項記載の核酸抽出デバイス。 The nucleic acid extraction device according to any one of claims 1 to 8, wherein a nucleic acid trapping portion is provided immediately below the cell trapping portion.
  10. 　前記細胞トラッピング部の直下以外の領域に核酸トラッピング部を設けたことを特徴とする請求項１～８のいずれか１項記載の核酸抽出デバイス。 The nucleic acid extraction device according to any one of claims 1 to 8, wherein a nucleic acid trapping portion is provided in a region other than immediately below the cell trapping portion.
  11. 　請求項１～１０のいずれか１項記載の核酸抽出デバイスと、cDNAライブラリを構築するための試薬を導入する手段とを有することを特徴とする核酸処理装置。 A nucleic acid processing apparatus comprising the nucleic acid extraction device according to any one of claims 1 to 10 and means for introducing a reagent for constructing a cDNA library.
  12. 　請求項１～１０のいずれか１項記載の核酸抽出デバイスと、cDNAライブラリを構築するための試薬と核酸増幅のための試薬を導入する手段とを有することを特徴とする核酸処理装置。 A nucleic acid processing apparatus comprising the nucleic acid extraction device according to any one of claims 1 to 10, a reagent for constructing a cDNA library, and a means for introducing a reagent for nucleic acid amplification.
  13. 　請求項１～１０のいずれか１項記載の核酸抽出デバイスと、細胞トラッピング部にトラップした細胞を微分干渉顕微鏡、位相差顕微鏡、ラマン顕微鏡またはコヒーレントラマン顕微鏡で観察するための顕微鏡部とを有することを特徴とする核酸処理装置。 A nucleic acid extraction device according to any one of claims 1 to 10, and a microscope unit for observing the cells trapped in the cell trapping unit with a differential interference microscope, a phase contrast microscope, a Raman microscope, or a coherent Raman microscope. A nucleic acid processing apparatus characterized by the above.
  14. 　細胞トラッピング部と、前記細胞トラッピング部より下方に配置される核酸トラッピング部とを備えた核酸抽出デバイスにより、細胞から核酸を抽出する方法であって、
    　前記細胞トラッピング部に細胞を接触させて、それぞれ１つの細胞を前記細胞トラッピング部にトラップする工程と、
    　細胞から核酸を抽出するための核酸抽出液を、前記細胞トラッピング部を通過して上から下へ通じる流路を通して流す工程と、
    　前記核酸トラッピング部に抽出された核酸を固定する工程と、
    　核酸抽出後の溶液を、前記核酸トラッピング部から前記細胞トラッピング部とは反対側に流路を介して排出する工程とを含み、
    　前記核酸抽出デバイスにおいて、前記細胞トラッピング部と、前記２つの流路と、前記核酸トラッピング部が上下方向に対を形成し、この対が、平面方向に複数配置されていることを特徴とする前記方法。     A method for extracting nucleic acid from a cell using a nucleic acid extraction device comprising a cell trapping unit and a nucleic acid trapping unit disposed below the cell trapping unit,
    Contacting a cell with the cell trapping portion and trapping each one cell in the cell trapping portion;
    Flowing a nucleic acid extract for extracting nucleic acid from cells through a flow path passing through the cell trapping section from top to bottom;
    Immobilizing the extracted nucleic acid in the nucleic acid trapping portion;
    Discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section through a flow path,
    In the nucleic acid extraction device, the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction. Method.
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